rowid,run.accession,experiment.accession,sample.accession,study.accession,bioproject,study.title,study.alias,study.type,study.abstract,study.attributes,study.PMIDs,sample.description,sample.title,sample.alias,sample.centername,sample.attributes,GEOsample.title,GEOsample.dataprocessing,GEOsample.source,GEOsample.treatmentprotocol,GEOsample.extractprotocol,GEOsample.growthprotocol,GEOsample.characteristics,GEOsample.accession,experiment.title,experiment.alias,experiment.library_name,experiment.design_description,experiment.library_construction_protocol,experiment.attributes,experiment.library_strategy,experiment.library_source,experiment.library_selection,experiment.library_layout,experiment.platform,experiment.instrument_model,experiment.spot_descriptor,experiment.study_ref,run.title,run.attributes,run.filename,run.semantic_name,run.total_bases,run.total_spots,run.alias,run.read_lengths,run.base_counts,run.r1_length,run.r2_length,run.r3_length,run.r4_length,run.Acount,run.Ccount,run.Gcount,run.Tcount,run.Ncount,run.experiment,run.pool_member,submission.accession,submission.srasource,submission.bioprojectsource,seqdetective.n_mates,seqdetective.mapping_rate.mate1,seqdetective.mapping_rate.mate2,seqdetective.nofeature_rate.mate1,seqdetective.nofeature_rate.mate2,seqdetective.sparsity.mate1,seqdetective.sparsity.mate2,seqdetective.pos_strand_rate.mate1,seqdetective.pos_strand_rate.mate2,seqdetective.readlen.mate1,seqdetective.readlen.mate2,seqdetective.judgement.mate1,seqdetective.judgement.mate2,seqdetective.judgement.reason,platform_family,instrument_generation,read_bias,selection_class,prep_kit,sc_or_bulk,tech_class,technology,tech_variant,submission.bioprojectsource.country,earliest_date,devstage_curation,devstage_curation_coarse,tissue_curation,tissue_curation_coarse
76740,SRR25374386,SRX21113512,SRS18382106,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 A10,GSM7655684,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 A10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655684,GSM7655684: ExVi2Red 01 A10; Danio rerio; RNA Seq,GSM7655684 r1,GSM7655684,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21286_Track-51602_R1.fastq.gz,fastq,38304684.0,504009.0,GSM7655684 r1,0:76,A:10444153;C:8397210;G:8505443;T:10956830;N:1048,76,,,,10444153,8397210,8505443,10956830,1048,SRX21113512,SRS18382106,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.78167,,0.12085,,0.94621,,0.44883,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76741,SRR25374387,SRX21113511,SRS18382105,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 B10,GSM7655683,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 B10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655683,GSM7655683: ExVi2Red 01 B10; Danio rerio; RNA Seq,GSM7655683 r1,GSM7655683,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21287_Track-51603_R1.fastq.gz,fastq,41818544.0,550244.0,GSM7655683 r1,0:76,A:11263718;C:9415229;G:9551199;T:11587507;N:891,76,,,,11263718,9415229,9551199,11587507,891,SRX21113511,SRS18382105,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.72269,,0.12564,,0.9542,,0.55822,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76742,SRR25374388,SRX21113510,SRS18382104,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 C10,GSM7655682,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 C10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655682,GSM7655682: ExVi2Red 01 C10; Danio rerio; RNA Seq,GSM7655682 r1,GSM7655682,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21288_Track-51604_R1.fastq.gz,fastq,41119648.0,541048.0,GSM7655682 r1,0:76,A:10732270;C:9662921;G:9639944;T:11083600;N:913,76,,,,10732270,9662921,9639944,11083600,913,SRX21113510,SRS18382104,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86963,,0.06809,,0.93292,,0.65489,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76743,SRR25374389,SRX21113509,SRS18382103,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 D10,GSM7655681,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 D10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655681,GSM7655681: ExVi2Red 01 D10; Danio rerio; RNA Seq,GSM7655681 r1,GSM7655681,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21289_Track-51605_R1.fastq.gz,fastq,42475108.0,558883.0,GSM7655681 r1,0:76,A:11042198;C:9997401;G:9991867;T:11442635;N:1007,76,,,,11042198,9997401,9991867,11442635,1007,SRX21113509,SRS18382103,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85259,,0.05736,,0.94223,,0.68549,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76744,SRR25374390,SRX21113508,SRS18382102,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 E10,GSM7655680,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 E10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655680,GSM7655680: ExVi2Red 01 E10; Danio rerio; RNA Seq,GSM7655680 r1,GSM7655680,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21290_Track-51606_R1.fastq.gz,fastq,42190564.0,555139.0,GSM7655680 r1,0:76,A:10880720;C:9985936;G:9919117;T:11403588;N:1203,76,,,,10880720,9985936,9919117,11403588,1203,SRX21113508,SRS18382102,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84883,,0.06243,,0.94387,,0.71713,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76745,SRR25374391,SRX21113507,SRS18382101,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 F10,GSM7655679,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 F10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655679,GSM7655679: ExVi2Red 01 F10; Danio rerio; RNA Seq,GSM7655679 r1,GSM7655679,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21291_Track-51607_R1.fastq.gz,fastq,30276196.0,398371.0,GSM7655679 r1,0:76,A:7966868;C:7016730;G:7000707;T:8291155;N:736,76,,,,7966868,7016730,7000707,8291155,736,SRX21113507,SRS18382101,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83303,,0.05427,,0.93785,,0.69443,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76746,SRR25374392,SRX21113506,SRS18382100,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 G10,GSM7655678,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 G10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655678,GSM7655678: ExVi2Red 01 G10; Danio rerio; RNA Seq,GSM7655678 r1,GSM7655678,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21292_Track-51608_R1.fastq.gz,fastq,42697028.0,561803.0,GSM7655678 r1,0:76,A:11421256;C:9416456;G:9762806;T:12095440;N:1070,76,,,,11421256,9416456,9762806,12095440,1070,SRX21113506,SRS18382100,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.4772,,0.03926,,0.94742,,0.64918,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76747,SRR25374393,SRX21113505,SRS18382099,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 H01,GSM7655653,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 H01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655653,GSM7655653: ExVi16Gr 02 H01; Danio rerio; RNA Seq,GSM7655653 r1,GSM7655653,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21317_Track-51633_R1.fastq.gz,fastq,39867624.0,524574.0,GSM7655653 r1,0:76,A:10664168;C:9072852;G:9105099;T:11024563;N:942,76,,,,10664168,9072852,9105099,11024563,942,SRX21113505,SRS18382099,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83797,,0.06671,,0.94044,,0.62345,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76748,SRR25374394,SRX21113504,SRS18382098,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 A02,GSM7655652,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 A02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655652,GSM7655652: ExVi16Gr 02 A02; Danio rerio; RNA Seq,GSM7655652 r1,GSM7655652,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21318_Track-51634_R1.fastq.gz,fastq,37836752.0,497852.0,GSM7655652 r1,0:76,A:10538160;C:8088084;G:8235430;T:10974153;N:925,76,,,,10538160,8088084,8235430,10974153,925,SRX21113504,SRS18382098,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.80039,,0.2249,,0.95392,,0.68704,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76749,SRR25374395,SRX21113503,SRS18382097,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 B02,GSM7655651,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 B02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655651,GSM7655651: ExVi16Gr 02 B02; Danio rerio; RNA Seq,GSM7655651 r1,GSM7655651,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21319_Track-51635_R1.fastq.gz,fastq,37765464.0,496914.0,GSM7655651 r1,0:76,A:10327605;C:8440804;G:8513957;T:10482149;N:949,76,,,,10327605,8440804,8513957,10482149,949,SRX21113503,SRS18382097,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.79171,,0.10658,,0.93785,,0.57458,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76750,SRR25374396,SRX21113502,SRS18382096,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 C02,GSM7655650,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 C02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655650,GSM7655650: ExVi16Gr 02 C02; Danio rerio; RNA Seq,GSM7655650 r1,GSM7655650,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21320_Track-51636_R1.fastq.gz,fastq,33616016.0,442316.0,GSM7655650 r1,0:76,A:9130691;C:7426220;G:7511228;T:9547023;N:854,76,,,,9130691,7426220,7511228,9547023,854,SRX21113502,SRS18382096,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.8161,,0.16087,,0.94282,,0.74593,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76751,SRR25374397,SRX21113501,SRS18382094,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 D02,GSM7655649,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 D02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655649,GSM7655649: ExVi16Gr 02 D02; Danio rerio; RNA Seq,GSM7655649 r1,GSM7655649,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21321_Track-51637_R1.fastq.gz,fastq,48530712.0,638562.0,GSM7655649 r1,0:76,A:12706296;C:11286685;G:11268611;T:13267981;N:1139,76,,,,12706296,11286685,11268611,13267981,1139,SRX21113501,SRS18382094,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87539,,0.05636,,0.94509,,0.67319,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76752,SRR25374398,SRX21113500,SRS18382095,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 E02,GSM7655648,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 E02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655648,GSM7655648: ExVi16Gr 02 E02; Danio rerio; RNA Seq,GSM7655648 r1,GSM7655648,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21322_Track-51638_R1.fastq.gz,fastq,38063080.0,500830.0,GSM7655648 r1,0:76,A:10345224;C:8528904;G:8613676;T:10574273;N:1003,76,,,,10345224,8528904,8613676,10574273,1003,SRX21113500,SRS18382095,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82962,,0.12581,,0.94334,,0.5417,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76753,SRR25374399,SRX21113499,SRS18382093,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 F02,GSM7655647,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 F02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655647,GSM7655647: ExVi16Gr 02 F02; Danio rerio; RNA Seq,GSM7655647 r1,GSM7655647,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21323_Track-51639_R1.fastq.gz,fastq,33778504.0,444454.0,GSM7655647 r1,0:76,A:8715964;C:7998179;G:7952604;T:9110906;N:851,76,,,,8715964,7998179,7952604,9110906,851,SRX21113499,SRS18382093,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86888,,0.04769,,0.9417,,0.71664,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76754,SRR25374400,SRX21113498,SRS18382091,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 G02,GSM7655646,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 G02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655646,GSM7655646: ExVi16Gr 02 G02; Danio rerio; RNA Seq,GSM7655646 r1,GSM7655646,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21324_Track-51640_R1.fastq.gz,fastq,38434264.0,505714.0,GSM7655646 r1,0:76,A:10173498;C:8859852;G:8871249;T:10528740;N:925,76,,,,10173498,8859852,8871249,10528740,925,SRX21113498,SRS18382091,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84613,,0.07553,,0.93839,,0.68257,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76755,SRR25374401,SRX21113497,SRS18382092,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 H04,GSM7655629,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 H04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655629,GSM7655629: ExVi16Gr 02 H04; Danio rerio; RNA Seq,GSM7655629 r1,GSM7655629,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21341_Track-51657_R1.fastq.gz,fastq,30968860.0,407485.0,GSM7655629 r1,0:76,A:8289373;C:7104056;G:7130406;T:8444292;N:733,76,,,,8289373,7104056,7130406,8444292,733,SRX21113497,SRS18382092,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.89416,,0.07736,,0.9217,,0.52336,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76756,SRR25374402,SRX21113496,SRS18382090,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 A05,GSM7655628,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 A05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655628,GSM7655628: ExVi16Gr 02 A05; Danio rerio; RNA Seq,GSM7655628 r1,GSM7655628,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21342_Track-51658_R1.fastq.gz,fastq,77775132.0,1023357.0,GSM7655628 r1,0:76,A:20285912;C:17896974;G:18105934;T:21484315;N:1997,76,,,,20285912,17896974,18105934,21484315,1997,SRX21113496,SRS18382090,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85173,,0.08581,,0.94923,,0.6598,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76757,SRR25374403,SRX21113495,SRS18382089,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 B05,GSM7655627,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 B05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655627,GSM7655627: ExVi16Gr 02 B05; Danio rerio; RNA Seq,GSM7655627 r1,GSM7655627,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21343_Track-51659_R1.fastq.gz,fastq,48071824.0,632524.0,GSM7655627 r1,0:76,A:13026880;C:10835674;G:10943843;T:13264257;N:1170,76,,,,13026880,10835674,10943843,13264257,1170,SRX21113495,SRS18382089,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.8831,,0.07207,,0.91881,,0.53229,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76758,SRR25374404,SRX21113494,SRS18382088,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 C05,GSM7655626,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 C05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655626,GSM7655626: ExVi16Gr 02 C05; Danio rerio; RNA Seq,GSM7655626 r1,GSM7655626,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21344_Track-51660_R1.fastq.gz,fastq,44401404.0,584229.0,GSM7655626 r1,0:76,A:11837676;C:10159931;G:10224645;T:12177927;N:1225,76,,,,11837676,10159931,10224645,12177927,1225,SRX21113494,SRS18382088,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.8662,,0.10475,,0.93939,,0.61495,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76759,SRR25374405,SRX21113493,SRS18382087,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 D05,GSM7655625,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 D05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655625,GSM7655625: ExVi16Gr 02 D05; Danio rerio; RNA Seq,GSM7655625 r1,GSM7655625,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21345_Track-51661_R1.fastq.gz,fastq,40097980.0,527605.0,GSM7655625 r1,0:76,A:10996482;C:8953433;G:9037603;T:11109378;N:1084,76,,,,10996482,8953433,9037603,11109378,1084,SRX21113493,SRS18382087,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.8426,,0.13642,,0.94245,,0.59961,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76760,SRR25374406,SRX21113492,SRS18382086,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,UHR 02 E05,GSM7655624,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,UHR 02 E05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655624,GSM7655624: UHR 02 E05; Danio rerio; RNA Seq,GSM7655624 r1,GSM7655624,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21346_Track-51662_R1.fastq.gz,fastq,73001572.0,960547.0,GSM7655624 r1,0:76,A:18801430;C:17125045;G:17615098;T:19458134;N:1865,76,,,,18801430,17125045,17615098,19458134,1865,SRX21113492,SRS18382086,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.00277,,0.00188,,0.99906,,0.73509,,76,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76761,SRR25374407,SRX21113491,SRS18382085,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 F05,GSM7655623,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 F05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655623,GSM7655623: ExVi16Gr 02 F05; Danio rerio; RNA Seq,GSM7655623 r1,GSM7655623,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21347_Track-51663_R1.fastq.gz,fastq,27670764.0,364089.0,GSM7655623 r1,0:76,A:7066830;C:6632931;G:6548167;T:7422248;N:588,76,,,,7066830,6632931,6548167,7422248,588,SRX21113491,SRS18382085,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87564,,0.03905,,0.92614,,0.74611,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76762,SRR25374408,SRX21113490,SRS18382083,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 G05,GSM7655622,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 G05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655622,GSM7655622: ExVi16Gr 02 G05; Danio rerio; RNA Seq,GSM7655622 r1,GSM7655622,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21348_Track-51664_R1.fastq.gz,fastq,42969260.0,565385.0,GSM7655622 r1,0:76,A:11590827;C:9713842;G:9805929;T:11857589;N:1073,76,,,,11590827,9713842,9805929,11857589,1073,SRX21113490,SRS18382083,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84464,,0.11633,,0.94939,,0.6516,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76763,SRR25374409,SRX21113489,SRS18382084,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 H08,GSM7655597,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 H08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655597,GSM7655597: ExVi16Gr 02 H08; Danio rerio; RNA Seq,GSM7655597 r1,GSM7655597,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21373_Track-51689_R1.fastq.gz,fastq,38007448.0,500098.0,GSM7655597 r1,0:76,A:10173426;C:8641625;G:8730744;T:10460660;N:993,76,,,,10173426,8641625,8730744,10460660,993,SRX21113489,SRS18382084,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.79669,,0.08276,,0.95067,,0.65876,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76764,SRR25374410,SRX21113488,SRS18382082,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 A09,GSM7655596,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 A09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655596,GSM7655596: ExVi16Gr 02 A09; Danio rerio; RNA Seq,GSM7655596 r1,GSM7655596,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21374_Track-51690_R1.fastq.gz,fastq,44346608.0,583508.0,GSM7655596 r1,0:76,A:11794284;C:10211396;G:10275551;T:12064327;N:1050,76,,,,11794284,10211396,10275551,12064327,1050,SRX21113488,SRS18382082,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84786,,0.10747,,0.9432,,0.64267,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76765,SRR25374411,SRX21113487,SRS18382081,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 B09,GSM7655595,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 B09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655595,GSM7655595: ExVi16Gr 02 B09; Danio rerio; RNA Seq,GSM7655595 r1,GSM7655595,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21375_Track-51691_R1.fastq.gz,fastq,43404512.0,571112.0,GSM7655595 r1,0:76,A:11406215;C:10089747;G:10096444;T:11811131;N:975,76,,,,11406215,10089747,10096444,11811131,975,SRX21113487,SRS18382081,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87723,,0.08948,,0.94365,,0.60874,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76766,SRR25374412,SRX21113486,SRS18382080,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 C09,GSM7655594,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 C09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655594,GSM7655594: ExVi16Gr 02 C09; Danio rerio; RNA Seq,GSM7655594 r1,GSM7655594,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21376_Track-51692_R1.fastq.gz,fastq,45027036.0,592461.0,GSM7655594 r1,0:76,A:11841919;C:10492780;G:10537256;T:12153887;N:1194,76,,,,11841919,10492780,10537256,12153887,1194,SRX21113486,SRS18382080,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84983,,0.08729,,0.93584,,0.63209,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76767,SRR25374413,SRX21113485,SRS18382079,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 D09,GSM7655593,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 D09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655593,GSM7655593: ExVi16Gr 02 D09; Danio rerio; RNA Seq,GSM7655593 r1,GSM7655593,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21377_Track-51693_R1.fastq.gz,fastq,12382908.0,162933.0,GSM7655593 r1,0:76,A:3238636;C:2869308;G:2881983;T:3392821;N:160,76,,,,3238636,2869308,2881983,3392821,160,SRX21113485,SRS18382079,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83061,,0.05654,,0.94558,,0.64901,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76768,SRR25374414,SRX21113484,SRS18382077,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 E09,GSM7655592,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 E09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655592,GSM7655592: ExVi16Gr 02 E09; Danio rerio; RNA Seq,GSM7655592 r1,GSM7655592,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21378_Track-51694_R1.fastq.gz,fastq,35251232.0,463832.0,GSM7655592 r1,0:76,A:9503812;C:8037895;G:8131353;T:9577315;N:857,76,,,,9503812,8037895,8131353,9577315,857,SRX21113484,SRS18382077,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.77275,,0.07657,,0.94836,,0.59607,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76769,SRR25374415,SRX21113483,SRS18382078,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 F09,GSM7655591,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 F09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655591,GSM7655591: ExVi16Gr 02 F09; Danio rerio; RNA Seq,GSM7655591 r1,GSM7655591,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21379_Track-51695_R1.fastq.gz,fastq,29051836.0,382261.0,GSM7655591 r1,0:76,A:7518130;C:6891176;G:6876887;T:7764943;N:700,76,,,,7518130,6891176,6876887,7764943,700,SRX21113483,SRS18382078,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86709,,0.07623,,0.94495,,0.65172,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76770,SRR25374416,SRX21113482,SRS18382076,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 G09,GSM7655590,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 G09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655590,GSM7655590: ExVi16Gr 02 G09; Danio rerio; RNA Seq,GSM7655590 r1,GSM7655590,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21380_Track-51696_R1.fastq.gz,fastq,109516.0,1441.0,GSM7655590 r1,0:76,A:29131;C:25838;G:25349;T:29198;N:0,76,,,,29131,25838,25349,29198,0,SRX21113482,SRS18382076,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.76586,,0.05934,,0.99277,,0.74208,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76771,SRR25374417,SRX21113481,SRS18382075,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 A01,GSM7655755,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 A01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655755,GSM7655755: ExVi2Red 01 A01; Danio rerio; RNA Seq,GSM7655755 r1,GSM7655755,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21214_Track-51531_R1.fastq.gz,fastq,45532968.0,599118.0,GSM7655755 r1,0:76,A:12246803;C:10349678;G:10361865;T:12573403;N:1219,76,,,,12246803,10349678,10361865,12573403,1219,SRX21113481,SRS18382075,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87285,,0.0881,,0.90136,,0.6883,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76772,SRR25374418,SRX21113480,SRS18382074,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 B01,GSM7655754,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 B01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655754,GSM7655754: ExVi2Red 01 B01; Danio rerio; RNA Seq,GSM7655754 r1,GSM7655754,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21215_Track-51532_R1.fastq.gz,fastq,34996100.0,460475.0,GSM7655754 r1,0:76,A:9331462;C:8037189;G:8071185;T:9555393;N:871,76,,,,9331462,8037189,8071185,9555393,871,SRX21113480,SRS18382074,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84607,,0.05475,,0.92374,,0.60845,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76773,SRR25374419,SRX21113479,SRS18382073,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 C01,GSM7655753,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 C01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655753,GSM7655753: ExVi2Red 01 C01; Danio rerio; RNA Seq,GSM7655753 r1,GSM7655753,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21216_Track-51533_R1.fastq.gz,fastq,35915548.0,472573.0,GSM7655753 r1,0:76,A:9876241;C:7948709;G:8031565;T:10058067;N:966,76,,,,9876241,7948709,8031565,10058067,966,SRX21113479,SRS18382073,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83728,,0.10675,,0.94426,,0.70513,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76774,SRR25374420,SRX21113478,SRS18382072,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 D01,GSM7655752,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 D01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655752,GSM7655752: ExVi2Red 01 D01; Danio rerio; RNA Seq,GSM7655752 r1,GSM7655752,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21217_Track-51534_R1.fastq.gz,fastq,40427896.0,531946.0,GSM7655752 r1,0:76,A:10795650;C:9203028;G:9208037;T:11220185;N:996,76,,,,10795650,9203028,9208037,11220185,996,SRX21113478,SRS18382072,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82317,,0.05906,,0.93229,,0.65906,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76775,SRR25374421,SRX21113477,SRS18382071,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 E01,GSM7655751,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 E01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655751,GSM7655751: ExVi2Red 01 E01; Danio rerio; RNA Seq,GSM7655751 r1,GSM7655751,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21218_Track-51535_R1.fastq.gz,fastq,44180016.0,581316.0,GSM7655751 r1,0:76,A:11920819;C:10035364;G:10070579;T:12151996;N:1258,76,,,,11920819,10035364,10070579,12151996,1258,SRX21113477,SRS18382071,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87211,,0.08161,,0.9011,,0.58341,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76776,SRR25374422,SRX21113476,SRS18382069,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 F01,GSM7655750,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 F01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655750,GSM7655750: ExVi2Red 01 F01; Danio rerio; RNA Seq,GSM7655750 r1,GSM7655750,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21219_Track-51536_R1.fastq.gz,fastq,33634636.0,442561.0,GSM7655750 r1,0:76,A:8955856;C:7717906;G:7739888;T:9220366;N:620,76,,,,8955856,7717906,7739888,9220366,620,SRX21113476,SRS18382069,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.79266,,0.0799,,0.93852,,0.6609,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76777,SRR25374423,SRX21113475,SRS18382070,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 G04,GSM7655725,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 G04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655725,GSM7655725: ExVi2Red 01 G04; Danio rerio; RNA Seq,GSM7655725 r1,GSM7655725,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21244_Track-51561_R1.fastq.gz,fastq,28279068.0,372093.0,GSM7655725 r1,0:76,A:7573141;C:6458500;G:6502179;T:7744670;N:578,76,,,,7573141,6458500,6502179,7744670,578,SRX21113475,SRS18382070,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82931,,0.07538,,0.93892,,0.57086,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76778,SRR25374424,SRX21113474,SRS18382068,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 H04,GSM7655724,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 H04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655724,GSM7655724: ExVi2Red 01 H04; Danio rerio; RNA Seq,GSM7655724 r1,GSM7655724,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21245_Track-51562_R1.fastq.gz,fastq,33646188.0,442713.0,GSM7655724 r1,0:76,A:8914312;C:7724087;G:7709961;T:9296909;N:919,76,,,,8914312,7724087,7709961,9296909,919,SRX21113474,SRS18382068,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83628,,0.07615,,0.94123,,0.62167,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76779,SRR25374425,SRX21113473,SRS18382067,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 A05,GSM7655723,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 A05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655723,GSM7655723: ExVi2Red 01 A05; Danio rerio; RNA Seq,GSM7655723 r1,GSM7655723,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21246_Track-51563_R1.fastq.gz,fastq,65374820.0,860195.0,GSM7655723 r1,0:76,A:17920811;C:14416412;G:14755021;T:18280774;N:1802,76,,,,17920811,14416412,14755021,18280774,1802,SRX21113473,SRS18382067,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82654,,0.12154,,0.93517,,0.62121,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76780,SRR25374426,SRX21113472,SRS18382066,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 B05,GSM7655722,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 B05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655722,GSM7655722: ExVi2Red 01 B05; Danio rerio; RNA Seq,GSM7655722 r1,GSM7655722,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21247_Track-51564_R1.fastq.gz,fastq,39210908.0,515933.0,GSM7655722 r1,0:76,A:10287821;C:9187861;G:9206063;T:10528196;N:967,76,,,,10287821,9187861,9206063,10528196,967,SRX21113472,SRS18382066,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86501,,0.0627,,0.91007,,0.61738,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76781,SRR25374427,SRX21113471,SRS18382065,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 C05,GSM7655721,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 C05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655721,GSM7655721: ExVi2Red 01 C05; Danio rerio; RNA Seq,GSM7655721 r1,GSM7655721,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21248_Track-51565_R1.fastq.gz,fastq,38625252.0,508227.0,GSM7655721 r1,0:76,A:10080873;C:9030644;G:8994423;T:10518327;N:985,76,,,,10080873,9030644,8994423,10518327,985,SRX21113471,SRS18382065,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86695,,0.06747,,0.93837,,0.67272,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76782,SRR25374428,SRX21113470,SRS18382063,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 D05,GSM7655720,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 D05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655720,GSM7655720: ExVi2Red 01 D05; Danio rerio; RNA Seq,GSM7655720 r1,GSM7655720,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21249_Track-51566_R1.fastq.gz,fastq,38599488.0,507888.0,GSM7655720 r1,0:76,A:10171275;C:8965675;G:8953753;T:10507855;N:930,76,,,,10171275,8965675,8953753,10507855,930,SRX21113470,SRS18382063,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84344,,0.069,,0.93519,,0.66364,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76783,SRR25374429,SRX21113469,SRS18382064,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,UHR 01 E05,GSM7655719,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,UHR 01 E05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655719,GSM7655719: UHR 01 E05; Danio rerio; RNA Seq,GSM7655719 r1,GSM7655719,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21250_Track-51567_R1.fastq.gz,fastq,37809468.0,497493.0,GSM7655719 r1,0:76,A:9802568;C:8894462;G:9020819;T:10090613;N:1006,76,,,,9802568,8894462,9020819,10090613,1006,SRX21113469,SRS18382064,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.00309,,0.00234,,0.99894,,0.71653,,76,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76784,SRR25374430,SRX21113468,SRS18382062,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 F05,GSM7655718,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 F05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655718,GSM7655718: ExVi2Red 01 F05; Danio rerio; RNA Seq,GSM7655718 r1,GSM7655718,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21251_Track-51568_R1.fastq.gz,fastq,27209672.0,358022.0,GSM7655718 r1,0:76,A:7189506;C:6365576;G:6376421;T:7277585;N:584,76,,,,7189506,6365576,6376421,7277585,584,SRX21113468,SRS18382062,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.89002,,0.05915,,0.8869,,0.58584,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76785,SRR25374431,SRX21113467,SRS18382061,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 H08,GSM7655693,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 H08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655693,GSM7655693: ExVi2Red 01 H08; Danio rerio; RNA Seq,GSM7655693 r1,GSM7655693,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21277_Track-51593_R1.fastq.gz,fastq,35576816.0,468116.0,GSM7655693 r1,0:76,A:9738994;C:7920726;G:7964832;T:9951202;N:1062,76,,,,9738994,7920726,7964832,9951202,1062,SRX21113467,SRS18382061,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82033,,0.12428,,0.92837,,0.61179,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76786,SRR25374432,SRX21113466,SRS18382060,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 A09,GSM7655692,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 A09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655692,GSM7655692: ExVi2Red 01 A09; Danio rerio; RNA Seq,GSM7655692 r1,GSM7655692,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21278_Track-51594_R1.fastq.gz,fastq,50878200.0,669450.0,GSM7655692 r1,0:76,A:13254053;C:11944278;G:11914998;T:13763637;N:1234,76,,,,13254053,11944278,11914998,13763637,1234,SRX21113466,SRS18382060,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.8501,,0.05515,,0.94221,,0.6349,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76787,SRR25374433,SRX21113465,SRS18382058,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 B09,GSM7655691,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 B09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655691,GSM7655691: ExVi2Red 01 B09; Danio rerio; RNA Seq,GSM7655691 r1,GSM7655691,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21279_Track-51595_R1.fastq.gz,fastq,37781120.0,497120.0,GSM7655691 r1,0:76,A:9776300;C:8939889;G:8924638;T:10139202;N:1091,76,,,,9776300,8939889,8924638,10139202,1091,SRX21113465,SRS18382058,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86291,,0.05774,,0.95022,,0.66413,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76788,SRR25374434,SRX21113464,SRS18382059,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 C09,GSM7655690,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 C09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655690,GSM7655690: ExVi2Red 01 C09; Danio rerio; RNA Seq,GSM7655690 r1,GSM7655690,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21280_Track-51596_R1.fastq.gz,fastq,38501144.0,506594.0,GSM7655690 r1,0:76,A:10447266;C:8626827;G:8684790;T:10741285;N:976,76,,,,10447266,8626827,8684790,10741285,976,SRX21113464,SRS18382059,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82592,,0.08168,,0.95213,,0.62373,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76789,SRR25374435,SRX21113463,SRS18382057,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 D09,GSM7655689,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 D09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655689,GSM7655689: ExVi2Red 01 D09; Danio rerio; RNA Seq,GSM7655689 r1,GSM7655689,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21281_Track-51597_R1.fastq.gz,fastq,37118476.0,488401.0,GSM7655689 r1,0:76,A:9991639;C:8479533;G:8508978;T:10137419;N:907,76,,,,9991639,8479533,8508978,10137419,907,SRX21113463,SRS18382057,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85202,,0.06249,,0.94095,,0.56917,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76790,SRR25374436,SRX21113462,SRS18382055,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 E09,GSM7655688,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 E09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655688,GSM7655688: ExVi2Red 01 E09; Danio rerio; RNA Seq,GSM7655688 r1,GSM7655688,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21282_Track-51598_R1.fastq.gz,fastq,34346604.0,451929.0,GSM7655688 r1,0:76,A:9152214;C:7869775;G:7863371;T:9460406;N:838,76,,,,9152214,7869775,7863371,9460406,838,SRX21113462,SRS18382055,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83557,,0.07471,,0.94657,,0.66911,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76791,SRR25374437,SRX21113461,SRS18382056,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 F09,GSM7655687,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 F09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655687,GSM7655687: ExVi2Red 01 F09; Danio rerio; RNA Seq,GSM7655687 r1,GSM7655687,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21283_Track-51599_R1.fastq.gz,fastq,38042484.0,500559.0,GSM7655687 r1,0:76,A:10115031;C:8815862;G:8847514;T:10263187;N:890,76,,,,10115031,8815862,8847514,10263187,890,SRX21113461,SRS18382056,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.88055,,0.0673,,0.91354,,0.60568,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76792,SRR25374438,SRX21113460,SRS18382054,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 G09,GSM7655686,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 G09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655686,GSM7655686: ExVi2Red 01 G09; Danio rerio; RNA Seq,GSM7655686 r1,GSM7655686,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21284_Track-51600_R1.fastq.gz,fastq,39984968.0,526118.0,GSM7655686 r1,0:76,A:10493521;C:9293954;G:9265128;T:10931579;N:786,76,,,,10493521,9293954,9265128,10931579,786,SRX21113460,SRS18382054,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86059,,0.06872,,0.94501,,0.67013,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76793,SRR25374439,SRX21113459,SRS18382053,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 H12,GSM7655661,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 H12,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655661,GSM7655661: ExVi2Red 01 H12; Danio rerio; RNA Seq,GSM7655661 r1,GSM7655661,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21309_Track-51625_R1.fastq.gz,fastq,42457552.0,558652.0,GSM7655661 r1,0:76,A:11537651;C:9525737;G:9537176;T:11855919;N:1069,76,,,,11537651,9525737,9537176,11855919,1069,SRX21113459,SRS18382053,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84093,,0.07999,,0.94458,,0.51356,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76794,SRR25374440,SRX21113458,SRS18382052,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 A01,GSM7655660,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 A01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655660,GSM7655660: ExVi16Gr 02 A01; Danio rerio; RNA Seq,GSM7655660 r1,GSM7655660,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21310_Track-51626_R1.fastq.gz,fastq,43953384.0,578334.0,GSM7655660 r1,0:76,A:12164178;C:9594866;G:9736597;T:12456729;N:1014,76,,,,12164178,9594866,9736597,12456729,1014,SRX21113458,SRS18382052,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83108,,0.12766,,0.94734,,0.62985,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76795,SRR25374441,SRX21113457,SRS18382050,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 B01,GSM7655659,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 B01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655659,GSM7655659: ExVi16Gr 02 B01; Danio rerio; RNA Seq,GSM7655659 r1,GSM7655659,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21311_Track-51627_R1.fastq.gz,fastq,40255300.0,529675.0,GSM7655659 r1,0:76,A:11092819;C:8866166;G:9025334;T:11270055;N:926,76,,,,11092819,8866166,9025334,11270055,926,SRX21113457,SRS18382050,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82194,,0.10415,,0.91334,,0.64999,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76796,SRR25374442,SRX21113456,SRS18382051,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 C01,GSM7655658,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 C01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655658,GSM7655658: ExVi16Gr 02 C01; Danio rerio; RNA Seq,GSM7655658 r1,GSM7655658,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21312_Track-51628_R1.fastq.gz,fastq,41825688.0,550338.0,GSM7655658 r1,0:76,A:11387557;C:9423691;G:9495623;T:11517753;N:1064,76,,,,11387557,9423691,9495623,11517753,1064,SRX21113456,SRS18382051,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87975,,0.1084,,0.89929,,0.677,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76797,SRR25374443,SRX21113455,SRS18382049,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 D01,GSM7655657,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 D01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655657,GSM7655657: ExVi16Gr 02 D01; Danio rerio; RNA Seq,GSM7655657 r1,GSM7655657,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21313_Track-51629_R1.fastq.gz,fastq,42909068.0,564593.0,GSM7655657 r1,0:76,A:11136421;C:10051973;G:10018387;T:11701257;N:1030,76,,,,11136421,10051973,10018387,11701257,1030,SRX21113455,SRS18382049,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.8659,,0.06904,,0.93783,,0.74091,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76798,SRR25374444,SRX21113454,SRS18382048,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 E01,GSM7655656,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 E01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655656,GSM7655656: ExVi16Gr 02 E01; Danio rerio; RNA Seq,GSM7655656 r1,GSM7655656,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21314_Track-51630_R1.fastq.gz,fastq,48570460.0,639085.0,GSM7655656 r1,0:76,A:13718330;C:10285583;G:10590684;T:13974798;N:1065,76,,,,13718330,10285583,10590684,13974798,1065,SRX21113454,SRS18382048,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83913,,0.1658,,0.9259,,0.60732,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76799,SRR25374445,SRX21113453,SRS18382046,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 F01,GSM7655655,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 F01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655655,GSM7655655: ExVi16Gr 02 F01; Danio rerio; RNA Seq,GSM7655655 r1,GSM7655655,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21315_Track-51631_R1.fastq.gz,fastq,24799028.0,326303.0,GSM7655655 r1,0:76,A:6908938;C:5394213;G:5481848;T:7013319;N:710,76,,,,6908938,5394213,5481848,7013319,710,SRX21113453,SRS18382046,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.74745,,0.14891,,0.94892,,0.60299,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76800,SRR25374446,SRX21113452,SRS18382047,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 G01,GSM7655654,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 G01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655654,GSM7655654: ExVi16Gr 02 G01; Danio rerio; RNA Seq,GSM7655654 r1,GSM7655654,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21316_Track-51632_R1.fastq.gz,fastq,38830756.0,510931.0,GSM7655654 r1,0:76,A:10376192;C:8800006;G:8855469;T:10798226;N:863,76,,,,10376192,8800006,8855469,10798226,863,SRX21113452,SRS18382047,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82638,,0.10507,,0.94923,,0.6744,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76801,SRR25374447,SRX21113451,SRS18382045,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 H03,GSM7655637,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 H03,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655637,GSM7655637: ExVi16Gr 02 H03; Danio rerio; RNA Seq,GSM7655637 r1,GSM7655637,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21333_Track-51649_R1.fastq.gz,fastq,69587804.0,915629.0,GSM7655637 r1,0:76,A:18290214;C:15924817;G:16122636;T:19248579;N:1558,76,,,,18290214,15924817,16122636,19248579,1558,SRX21113451,SRS18382045,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86176,,0.05715,,0.9498,,0.66804,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76802,SRR25374448,SRX21113450,SRS18382043,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 A04,GSM7655636,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 A04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655636,GSM7655636: ExVi16Gr 02 A04; Danio rerio; RNA Seq,GSM7655636 r1,GSM7655636,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21334_Track-51650_R1.fastq.gz,fastq,48709160.0,640910.0,GSM7655636 r1,0:76,A:12671466;C:11358110;G:11311161;T:13367355;N:1068,76,,,,12671466,11358110,11311161,13367355,1068,SRX21113450,SRS18382043,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86689,,0.06245,,0.93472,,0.72431,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76803,SRR25374449,SRX21113449,SRS18382044,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 B04,GSM7655635,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 B04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655635,GSM7655635: ExVi16Gr 02 B04; Danio rerio; RNA Seq,GSM7655635 r1,GSM7655635,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21335_Track-51651_R1.fastq.gz,fastq,52290508.0,688033.0,GSM7655635 r1,0:76,A:14530326;C:11216288;G:11546134;T:14996462;N:1298,76,,,,14530326,11216288,11546134,14996462,1298,SRX21113449,SRS18382044,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83555,,0.1433,,0.9487,,0.64941,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76804,SRR25374450,SRX21113448,SRS18382042,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 C04,GSM7655634,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 C04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655634,GSM7655634: ExVi16Gr 02 C04; Danio rerio; RNA Seq,GSM7655634 r1,GSM7655634,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21336_Track-51652_R1.fastq.gz,fastq,39331824.0,517524.0,GSM7655634 r1,0:76,A:10721038;C:8801213;G:8883803;T:10924746;N:1024,76,,,,10721038,8801213,8883803,10924746,1024,SRX21113448,SRS18382042,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85756,,0.11615,,0.93257,,0.61606,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76805,SRR25374451,SRX21113447,SRS18382041,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 D04,GSM7655633,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 D04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655633,GSM7655633: ExVi16Gr 02 D04; Danio rerio; RNA Seq,GSM7655633 r1,GSM7655633,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21337_Track-51653_R1.fastq.gz,fastq,39195252.0,515727.0,GSM7655633 r1,0:76,A:10235765;C:9178220;G:9168229;T:10612124;N:914,76,,,,10235765,9178220,9168229,10612124,914,SRX21113447,SRS18382041,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87007,,0.0635,,0.93486,,0.67648,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76806,SRR25374452,SRX21113446,SRS18382040,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 E04,GSM7655632,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 E04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655632,GSM7655632: ExVi16Gr 02 E04; Danio rerio; RNA Seq,GSM7655632 r1,GSM7655632,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21338_Track-51654_R1.fastq.gz,fastq,37489660.0,493285.0,GSM7655632 r1,0:76,A:9953560;C:8657951;G:8680136;T:10197142;N:871,76,,,,9953560,8657951,8680136,10197142,871,SRX21113446,SRS18382040,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.88354,,0.0859,,0.92939,,0.67823,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76807,SRR25374453,SRX21113445,SRS18382039,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 F04,GSM7655631,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 F04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655631,GSM7655631: ExVi16Gr 02 F04; Danio rerio; RNA Seq,GSM7655631 r1,GSM7655631,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21339_Track-51655_R1.fastq.gz,fastq,19105640.0,251390.0,GSM7655631 r1,0:76,A:5236468;C:4255113;G:4300729;T:5312886;N:444,76,,,,5236468,4255113,4300729,5312886,444,SRX21113445,SRS18382039,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83796,,0.12304,,0.95047,,0.58146,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76808,SRR25374454,SRX21113444,SRS18382038,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 G04,GSM7655630,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 G04,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655630,GSM7655630: ExVi16Gr 02 G04; Danio rerio; RNA Seq,GSM7655630 r1,GSM7655630,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21340_Track-51656_R1.fastq.gz,fastq,30408588.0,400113.0,GSM7655630 r1,0:76,A:8167511;C:6899738;G:6963601;T:8377003;N:735,76,,,,8167511,6899738,6963601,8377003,735,SRX21113444,SRS18382038,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.8294,,0.11531,,0.94974,,0.55704,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76809,SRR25374455,SRX21113443,SRS18382037,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 H07,GSM7655605,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 H07,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655605,GSM7655605: ExVi16Gr 02 H07; Danio rerio; RNA Seq,GSM7655605 r1,GSM7655605,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21365_Track-51681_R1.fastq.gz,fastq,59328716.0,780641.0,GSM7655605 r1,0:76,A:16209508;C:13080597;G:13472000;T:16565113;N:1498,76,,,,16209508,13080597,13472000,16565113,1498,SRX21113443,SRS18382037,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82175,,0.10813,,0.95215,,0.62231,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76810,SRR25374456,SRX21113442,SRS18382036,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 A08,GSM7655604,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 A08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655604,GSM7655604: ExVi16Gr 02 A08; Danio rerio; RNA Seq,GSM7655604 r1,GSM7655604,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21366_Track-51682_R1.fastq.gz,fastq,51310792.0,675142.0,GSM7655604 r1,0:76,A:13900416;C:11593094;G:11721806;T:14094161;N:1315,76,,,,13900416,11593094,11721806,14094161,1315,SRX21113442,SRS18382036,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85836,,0.11644,,0.95187,,0.57501,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76811,SRR25374457,SRX21113441,SRS18382035,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 B08,GSM7655603,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 B08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655603,GSM7655603: ExVi16Gr 02 B08; Danio rerio; RNA Seq,GSM7655603 r1,GSM7655603,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21367_Track-51683_R1.fastq.gz,fastq,46582148.0,612923.0,GSM7655603 r1,0:76,A:12304654;C:10864221;G:10886753;T:12525402;N:1118,76,,,,12304654,10864221,10886753,12525402,1118,SRX21113441,SRS18382035,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.89312,,0.07352,,0.91433,,0.63388,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76812,SRR25374458,SRX21113440,SRS18382033,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 C08,GSM7655602,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 C08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655602,GSM7655602: ExVi16Gr 02 C08; Danio rerio; RNA Seq,GSM7655602 r1,GSM7655602,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21368_Track-51684_R1.fastq.gz,fastq,35886364.0,472189.0,GSM7655602 r1,0:76,A:9500185;C:8254139;G:8296080;T:9835053;N:907,76,,,,9500185,8254139,8296080,9835053,907,SRX21113440,SRS18382033,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87376,,0.09726,,0.94815,,0.66337,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76813,SRR25374459,SRX21113439,SRS18382034,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 D08,GSM7655601,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 D08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655601,GSM7655601: ExVi16Gr 02 D08; Danio rerio; RNA Seq,GSM7655601 r1,GSM7655601,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21369_Track-51685_R1.fastq.gz,fastq,42306236.0,556661.0,GSM7655601 r1,0:76,A:11372293;C:9608044;G:9706729;T:11618141;N:1029,76,,,,11372293,9608044,9706729,11618141,1029,SRX21113439,SRS18382034,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84883,,0.10733,,0.94681,,0.64059,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76814,SRR25374460,SRX21113438,SRS18382032,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 E08,GSM7655600,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 E08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655600,GSM7655600: ExVi16Gr 02 E08; Danio rerio; RNA Seq,GSM7655600 r1,GSM7655600,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21370_Track-51686_R1.fastq.gz,fastq,39242752.0,516352.0,GSM7655600 r1,0:76,A:11005544;C:8404753;G:8635524;T:11195931;N:1000,76,,,,11005544,8404753,8635524,11195931,1000,SRX21113438,SRS18382032,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.79137,,0.21123,,0.94734,,0.50905,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76815,SRR25374461,SRX21113437,SRS18382031,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 F08,GSM7655599,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 F08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655599,GSM7655599: ExVi16Gr 02 F08; Danio rerio; RNA Seq,GSM7655599 r1,GSM7655599,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21371_Track-51687_R1.fastq.gz,fastq,25611544.0,336994.0,GSM7655599 r1,0:76,A:6885033;C:5851757;G:5912702;T:6961334;N:718,76,,,,6885033,5851757,5912702,6961334,718,SRX21113437,SRS18382031,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85629,,0.07491,,0.94612,,0.47807,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76816,SRR25374462,SRX21113436,SRS18382030,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 G08,GSM7655598,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 G08,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655598,GSM7655598: ExVi16Gr 02 G08; Danio rerio; RNA Seq,GSM7655598 r1,GSM7655598,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21372_Track-51688_R1.fastq.gz,fastq,31633784.0,416234.0,GSM7655598 r1,0:76,A:8424533;C:7308083;G:7375550;T:8524916;N:702,76,,,,8424533,7308083,7375550,8524916,702,SRX21113436,SRS18382030,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86078,,0.08815,,0.94339,,0.60325,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76817,SRR25374463,SRX21113435,SRS18382029,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 H09,GSM7655589,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 H09,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655589,GSM7655589: ExVi16Gr 02 H09; Danio rerio; RNA Seq,GSM7655589 r1,GSM7655589,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21381_Track-51697_R1.fastq.gz,fastq,46183528.0,607678.0,GSM7655589 r1,0:76,A:12578933;C:10422493;G:10545686;T:12635244;N:1172,76,,,,12578933,10422493,10545686,12635244,1172,SRX21113435,SRS18382029,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82775,,0.12894,,0.94554,,0.57796,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76818,SRR25374464,SRX21113434,SRS18382028,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 A10,GSM7655588,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 A10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655588,GSM7655588: ExVi16Gr 02 A10; Danio rerio; RNA Seq,GSM7655588 r1,GSM7655588,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21382_Track-51698_R1.fastq.gz,fastq,52786484.0,694559.0,GSM7655588 r1,0:76,A:13727831;C:12528209;G:12479195;T:14049800;N:1449,76,,,,13727831,12528209,12479195,14049800,1449,SRX21113434,SRS18382028,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.89619,,0.07198,,0.81728,,0.58493,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76819,SRR25374465,SRX21113433,SRS18382027,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 B10,GSM7655587,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 B10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655587,GSM7655587: ExVi16Gr 02 B10; Danio rerio; RNA Seq,GSM7655587 r1,GSM7655587,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21383_Track-51699_R1.fastq.gz,fastq,36607756.0,481681.0,GSM7655587 r1,0:76,A:9828936;C:8336713;G:8406059;T:10035219;N:829,76,,,,9828936,8336713,8406059,10035219,829,SRX21113433,SRS18382027,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83722,,0.0981,,0.94308,,0.59878,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76820,SRR25374466,SRX21113432,SRS18382026,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 C10,GSM7655586,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 C10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655586,GSM7655586: ExVi16Gr 02 C10; Danio rerio; RNA Seq,GSM7655586 r1,GSM7655586,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21384_Track-51700_R1.fastq.gz,fastq,40180516.0,528691.0,GSM7655586 r1,0:76,A:10915377;C:9054366;G:9171827;T:11038036;N:910,76,,,,10915377,9054366,9171827,11038036,910,SRX21113432,SRS18382026,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.80118,,0.13894,,0.94953,,0.50264,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76821,SRR25374467,SRX21113431,SRS18382025,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 D10,GSM7655585,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 D10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655585,GSM7655585: ExVi16Gr 02 D10; Danio rerio; RNA Seq,GSM7655585 r1,GSM7655585,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21385_Track-51701_R1.fastq.gz,fastq,42589488.0,560388.0,GSM7655585 r1,0:76,A:11369879;C:9826159;G:9905501;T:11486692;N:1257,76,,,,11369879,9826159,9905501,11486692,1257,SRX21113431,SRS18382025,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87025,,0.12385,,0.92072,,0.68164,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76822,SRR25374468,SRX21113430,SRS18382024,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 E10,GSM7655584,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 E10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655584,GSM7655584: ExVi16Gr 02 E10; Danio rerio; RNA Seq,GSM7655584 r1,GSM7655584,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21386_Track-51702_R1.fastq.gz,fastq,51684408.0,680058.0,GSM7655584 r1,0:76,A:14060182;C:11628397;G:11829324;T:14165366;N:1139,76,,,,14060182,11628397,11829324,14165366,1139,SRX21113430,SRS18382024,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83022,,0.10491,,0.9558,,0.6376,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76823,SRR25374469,SRX21113429,SRS18382023,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 F10,GSM7655583,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 F10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655583,GSM7655583: ExVi16Gr 02 F10; Danio rerio; RNA Seq,GSM7655583 r1,GSM7655583,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21387_Track-51703_R1.fastq.gz,fastq,36035248.0,474148.0,GSM7655583 r1,0:76,A:9424347;C:8412906;G:8429736;T:9767287;N:972,76,,,,9424347,8412906,8429736,9767287,972,SRX21113429,SRS18382023,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86746,,0.08476,,0.94477,,0.69679,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76824,SRR25374470,SRX21113428,SRS18382022,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi16Gr 02 G10,GSM7655582,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi16Gr 02 G10,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655582,GSM7655582: ExVi16Gr 02 G10; Danio rerio; RNA Seq,GSM7655582 r1,GSM7655582,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21388_Track-51704_R1.fastq.gz,fastq,49615992.0,652842.0,GSM7655582 r1,0:76,A:12837307;C:11724189;G:11709754;T:13343466;N:1276,76,,,,12837307,11724189,11709754,13343466,1276,SRX21113428,SRS18382022,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87302,,0.06776,,0.94217,,0.70686,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76825,SRR25374471,SRX21113427,SRS18382021,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 G03,GSM7655733,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 G03,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655733,GSM7655733: ExVi2Red 01 G03; Danio rerio; RNA Seq,GSM7655733 r1,GSM7655733,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21236_Track-51553_R1.fastq.gz,fastq,21310248.0,280398.0,GSM7655733 r1,0:76,A:5748959;C:4822220;G:4847200;T:5891460;N:409,76,,,,5748959,4822220,4847200,5891460,409,SRX21113427,SRS18382021,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87639,,0.10358,,0.91644,,0.5443,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76826,SRR25374472,SRX21113426,SRS18382020,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 H03,GSM7655732,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 H03,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655732,GSM7655732: ExVi2Red 01 H03; Danio rerio; RNA Seq,GSM7655732 r1,GSM7655732,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21237_Track-51554_R1.fastq.gz,fastq,34727060.0,456935.0,GSM7655732 r1,0:76,A:9637319;C:7417768;G:7547169;T:10124016;N:788,76,,,,9637319,7417768,7547169,10124016,788,SRX21113426,SRS18382020,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.79009,,0.15338,,0.92176,,0.56495,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76827,SRR25374369,SRX21113425,SRS18382019,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 G01,GSM7655749,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 G01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655749,GSM7655749: ExVi2Red 01 G01; Danio rerio; RNA Seq,GSM7655749 r1,GSM7655749,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21220_Track-51537_R1.fastq.gz,fastq,43573308.0,573333.0,GSM7655749 r1,0:76,A:11729831;C:9844461;G:9894913;T:12103016;N:1087,76,,,,11729831,9844461,9894913,12103016,1087,SRX21113425,SRS18382019,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86107,,0.08254,,0.88422,,0.61762,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76828,SRR25374370,SRX21113424,SRS18382018,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 H01,GSM7655748,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 H01,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655748,GSM7655748: ExVi2Red 01 H01; Danio rerio; RNA Seq,GSM7655748 r1,GSM7655748,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21221_Track-51538_R1.fastq.gz,fastq,49813212.0,655437.0,GSM7655748 r1,0:76,A:13595353;C:11047705;G:11150211;T:14018632;N:1311,76,,,,13595353,11047705,11150211,14018632,1311,SRX21113424,SRS18382018,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85528,,0.09114,,0.8826,,0.58107,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76829,SRR25374371,SRX21113423,SRS18382016,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 A02,GSM7655747,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 A02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655747,GSM7655747: ExVi2Red 01 A02; Danio rerio; RNA Seq,GSM7655747 r1,GSM7655747,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21222_Track-51539_R1.fastq.gz,fastq,39303932.0,517157.0,GSM7655747 r1,0:76,A:10336389;C:9149126;G:9155287;T:10662086;N:1044,76,,,,10336389,9149126,9155287,10662086,1044,SRX21113423,SRS18382016,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87787,,0.0686,,0.92719,,0.62874,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76830,SRR25374372,SRX21113422,SRS18382017,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 B02,GSM7655746,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 B02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655746,GSM7655746: ExVi2Red 01 B02; Danio rerio; RNA Seq,GSM7655746 r1,GSM7655746,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21223_Track-51540_R1.fastq.gz,fastq,38886616.0,511666.0,GSM7655746 r1,0:76,A:10350384;C:9020188;G:9044941;T:10470162;N:941,76,,,,10350384,9020188,9044941,10470162,941,SRX21113422,SRS18382017,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.89328,,0.04461,,0.91149,,0.7077,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76831,SRR25374373,SRX21113421,SRS18382015,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 C02,GSM7655745,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 C02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655745,GSM7655745: ExVi2Red 01 C02; Danio rerio; RNA Seq,GSM7655745 r1,GSM7655745,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21224_Track-51541_R1.fastq.gz,fastq,36512072.0,480422.0,GSM7655745 r1,0:76,A:9647899;C:8450542;G:8469310;T:9943477;N:844,76,,,,9647899,8450542,8469310,9943477,844,SRX21113421,SRS18382015,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.82734,,0.07569,,0.95424,,0.68572,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76832,SRR25374374,SRX21113420,SRS18382014,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 D02,GSM7655744,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 D02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655744,GSM7655744: ExVi2Red 01 D02; Danio rerio; RNA Seq,GSM7655744 r1,GSM7655744,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21225_Track-51542_R1.fastq.gz,fastq,39534972.0,520197.0,GSM7655744 r1,0:76,A:10630574;C:9014117;G:9058281;T:10831132;N:868,76,,,,10630574,9014117,9058281,10831132,868,SRX21113420,SRS18382014,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.84999,,0.07505,,0.92725,,0.59058,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76833,SRR25374375,SRX21113419,SRS18382013,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 E02,GSM7655743,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 E02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655743,GSM7655743: ExVi2Red 01 E02; Danio rerio; RNA Seq,GSM7655743 r1,GSM7655743,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21226_Track-51543_R1.fastq.gz,fastq,41187440.0,541940.0,GSM7655743 r1,0:76,A:11154626;C:9344165;G:9412118;T:11275602;N:929,76,,,,11154626,9344165,9412118,11275602,929,SRX21113419,SRS18382013,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86042,,0.08694,,0.9177,,0.62968,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76834,SRR25374376,SRX21113418,SRS18382012,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 F02,GSM7655742,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 F02,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655742,GSM7655742: ExVi2Red 01 F02; Danio rerio; RNA Seq,GSM7655742 r1,GSM7655742,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21227_Track-51544_R1.fastq.gz,fastq,32821056.0,431856.0,GSM7655742 r1,0:76,A:8748724;C:7515948;G:7530033;T:9025431;N:920,76,,,,8748724,7515948,7530033,9025431,920,SRX21113418,SRS18382012,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85486,,0.06705,,0.92226,,0.62428,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76835,SRR25374377,SRX21113417,SRS18382011,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 G05,GSM7655717,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 G05,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655717,GSM7655717: ExVi2Red 01 G05; Danio rerio; RNA Seq,GSM7655717 r1,GSM7655717,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21252_Track-51569_R1.fastq.gz,fastq,35300176.0,464476.0,GSM7655717 r1,0:76,A:9213609;C:8131851;G:8175755;T:9778363;N:598,76,,,,9213609,8131851,8175755,9778363,598,SRX21113417,SRS18382011,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.85828,,0.04123,,0.93107,,0.68057,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76836,SRR25374378,SRX21113416,SRS18382010,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 A06,GSM7655716,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 A06,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655716,GSM7655716: ExVi2Red 01 A06; Danio rerio; RNA Seq,GSM7655716 r1,GSM7655716,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21254_Track-51570_R1.fastq.gz,fastq,42251972.0,555947.0,GSM7655716 r1,0:76,A:10996315;C:9911874;G:9827456;T:11515372;N:955,76,,,,10996315,9911874,9827456,11515372,955,SRX21113416,SRS18382010,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.86098,,0.06657,,0.93253,,0.71096,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76837,SRR25374379,SRX21113415,SRS18382009,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 B06,GSM7655715,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 B06,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655715,GSM7655715: ExVi2Red 01 B06; Danio rerio; RNA Seq,GSM7655715 r1,GSM7655715,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21255_Track-51571_R1.fastq.gz,fastq,31369836.0,412761.0,GSM7655715 r1,0:76,A:8294873;C:7282380;G:7298071;T:8493814;N:698,76,,,,8294873,7282380,7298071,8493814,698,SRX21113415,SRS18382009,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87982,,0.06734,,0.93357,,0.58825,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76838,SRR25374380,SRX21113414,SRS18382007,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 C06,GSM7655714,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 C06,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655714,GSM7655714: ExVi2Red 01 C06; Danio rerio; RNA Seq,GSM7655714 r1,GSM7655714,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21256_Track-51572_R1.fastq.gz,fastq,29075092.0,382567.0,GSM7655714 r1,0:76,A:7651510;C:6803630;G:6808818;T:7810307;N:827,76,,,,7651510,6803630,6808818,7810307,827,SRX21113414,SRS18382007,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.83731,,0.07054,,0.94464,,0.63184,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System
76839,SRR25374381,SRX21113413,SRS18382008,SRP450614,PRJNA996961,Single cell transcriptome profiling of zebrafish pancreatic beta cells segregated by CaMPARI based calcium labeling.,GSE237867,Transcriptome Analysis,Coordination of cell activity trough Ca2+ waves enables pancreatic ß cells to secrete precise quantities of insulin in response to blood sugar. However  how the coordinating Ca2+ waves are orchestrated in space and time remains unknown. By applying functional Ca2+ imaging  using CaMPARI and single molecule RNA detection  we reveal a gene signature depicting a molecularly and metabolically distinct state of first responder ß cells.  We highlight Vitamin B6 metabolism as a signature pathway associated with the first responder ß cells . Overall design: Ex vivo photoconversion of ins:CaMPARI islets and Fluorescence activated cell sorting FACS The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.  The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence.  Single cell RNA Sequencing For single cell RNA Seq  ß cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  ß cells were collected from two conditions: responsive ß cells displaying photoconversion at 2.5 mM; and non responsive ß cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5.,,pubmed:38924394,,ExVi2Red 01 D06,GSM7655713,,source name:pancreas|tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion|geo loc name:missing|collection date:missing,ExVi2Red 01 D06,Raw reads in fastq format were trimmed using trim galore with default parameters to remove adapter sequences.  Trimmed reads were aligned to the zebrafish genome  GRCz10  using HISAT2 with default parameters. htseq count was used to assign reads to exons thus eventually getting counts per gene. For analysis  cells with more than 800 detected genes were utilized.  Transcriptional profile of responsive and non responsive were compared using ROTS with default parameters.  Genes with false discovery rate FDR less than 0.05 were considered as differentially expressed. For gene ontology GO analysis of differentially expressed genes  FishEnrichR was utilized.  As background  genes expressed in at least 5 % of the cells were used. Assembly: GRCz10 Supplementary files format and content: csv,pancreas,The primary islets from 4 mpf Tgins:CaMPARI animals were dissected from the fish post euthanasia.  Each islet was placed in a well of 24 well plate with 5 ml of DPBS medium supplemented with calcium  magnesium ThermoFisher  14040141  along with the appropriate concentration of D Glucose.  The islets were allowed to equilibrate for 5 min.  Subsequently  the entire 24 well plate was photoconverted using a 405 nm LED array Loctite  1167593 for 3 seconds.  The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch.  An internal timer using an LED controller Loctite  1359255 was utilized for precise timing of light exposure.,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,4 mpf Tgins:CaMPARI zebrafish,tissue:pancreas|age:4 mpf|cell type:beta cell|genotype:Tgins:CaMPARI|treatment:photoconversion,GSM7655713,GSM7655713: ExVi2Red 01 D06; Danio rerio; RNA Seq,GSM7655713 r1,GSM7655713,1,The photoconverted islets were dissociated into single cells by incubation in TrypLE ThermoFisher  12563029 at 37°C in a benchtop shaker set at 450 rpm for 45 min.  Following dissociation  TrypLE was inactivated with 10% FBS  and the cells pelleted by centrifugation at 500 g for 10 min at 4°C.  The supernatant was carefully discarded  and the pellet re suspended in 500 µl of HBSS without xxx+  Mg2+.  To remove debris  the solution was passed over a 30 µm cell filter Miltenyi Biotec  130 041 407.  To remove dead cells  calcein violet ThermoFisher  C34858 was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes.  The single cell preparation was sorted through 100 µm nozzle.  The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC Fluorescein isothiocyanate for green fluorescence; and PE Phycoerythrin for red fluorescence. For single cell RNA Seq  β cells isolated from Tgins:CaMPARI islets were subjected to profiling using Smart Seq2 protocol from Takara.  For this  islets were isolated  photoconverted ex vivo  dissociated and FAC sorted as described above.  β cells were collected from two conditions: responsive β cells displaying photoconversion at 2.5 mM; and non responsive β cells without xxx at 16.5 mM.  Photoconversion was defined as a high red to green ratio.  For FACS of each condition  index sorting was utilized  and the cells were collected in 96 well plate containing lysis buffer 2 µl of nuclease free water with 0.2% Triton X 100 and 4 U murine RNase Inhibitor NEB.  For each condition a total of 93 cells were collected for profiling.  Preparation of cDNA library and sequencing was performed exactly as described in a previous publication Singh et al.  Scientific Reports  2018; doi: 10.1038/s41598 018 35218 5. Smart Seq2 from Takara,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP450614,,loader:fastq load.py,L21257_Track-51573_R1.fastq.gz,fastq,43700076.0,575001.0,GSM7655713 r1,0:76,A:11632997;C:10024534;G:10021867;T:12019640;N:1038,76,,,,11632997,10024534,10021867,12019640,1038,SRX21113413,SRS18382008,SRA1676813,"Regeneration and Stress Biology, IRIBHM, ULB","Regeneration and Stress Biology, IRIBHM, ULB",1,0.87398,,0.07234,,0.90725,,0.67622,,76,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Belgium,2023-07-20,Adult,Adult,Pancreas,Endocrine System