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
10060,ERR4795364,ERX4665135,ERS5281176,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 4,E MTAB 9727:Sample 4,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 4 s,Sample 4 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 4,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN4_AH2TW3BGX5_S1_R1_cat.fastq.gz,fastq,5410723202.0,71707309.0,E MTAB 9727:Sample 4,0:75.46 1:0,A:1330416803;C:531269504;G:734031980;T:2814959249;N:45666,75,0,,,1330416803,531269504,734031980,2814959249,45666,ERX4665135,ERS5281176,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.33614,,0.21532,,0.99019,,0.41002,,76,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
10061,ERR4795365,ERX4665135,ERS5281176,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 4,E MTAB 9727:Sample 4,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 4 s,Sample 4 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 4,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN4_AH2TW3BGX5_S1_R2_cat.fastq.gz,fastq,5414184547.0,71707309.0,E MTAB 9727:Sample 4 1,0:0 1:75.50,A:1582938104;C:1052565419;G:1177180395;T:1600161662;N:1338967,0,75,,,1582938104,1052565419,1177180395,1600161662,1338967,ERX4665135,ERS5281176,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.84976,,0.28521,,0.85038,,0.5124,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
10062,ERR4795362,ERX4665134,ERS5281175,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 3,E MTAB 9727:Sample 3,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 3 s,Sample 3 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 3,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN3_AHY3WGBGX3_S7_R1_cat.fastq.gz,fastq,4823193891.0,63965519.0,E MTAB 9727:Sample 3,0:75.40 1:0,A:1323978162;C:446831209;G:581746343;T:2470114091;N:524086,75,0,,,1323978162,446831209,581746343,2470114091,524086,ERX4665134,ERS5281175,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.36549,,0.19722,,0.95552,,0.4702,,76,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
10063,ERR4795363,ERX4665134,ERS5281175,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 3,E MTAB 9727:Sample 3,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 3 s,Sample 3 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 3,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN3_AHY3WGBGX3_S7_R2_cat.fastq.gz,fastq,4828889391.0,63965519.0,E MTAB 9727:Sample 3 1,0:0 1:75.49,A:1434099697;C:964508763;G:893584575;T:1534727600;N:1968756,0,75,,,1434099697,964508763,893584575,1534727600,1968756,ERX4665134,ERS5281175,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.85811,,0.26231,,0.82731,,0.48618,,76,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
10064,ERR4795360,ERX4665133,ERS5281174,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 2,E MTAB 9727:Sample 2,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 2 s,Sample 2 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 2,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN2_AHY3WGBGX3_S6_R1_cat.fastq.gz,fastq,5598371393.0,74235388.0,E MTAB 9727:Sample 2,0:75.41 1:0,A:1526486973;C:558020193;G:717587491;T:2795646081;N:630655,75,0,,,1526486973,558020193,717587491,2795646081,630655,ERX4665133,ERS5281174,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.28059,,0.19252,,0.96404,,0.4874,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
10065,ERR4795361,ERX4665133,ERS5281174,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 2,E MTAB 9727:Sample 2,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 2 s,Sample 2 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 2,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN2_AHY3WGBGX3_S6_R2_cat.fastq.gz,fastq,5600151371.0,74235388.0,E MTAB 9727:Sample 2 1,0:0 1:75.44,A:1850568176;C:1035622971;G:1071138281;T:1640475883;N:2346060,0,75,,,1850568176,1035622971,1071138281,1640475883,2346060,ERX4665133,ERS5281174,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.7679,,0.30371,,0.82651,,0.43401,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
10066,ERR4795358,ERX4665132,ERS5281173,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 1,E MTAB 9727:Sample 1,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 1 s,Sample 1 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 1,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN1_AHY3WGBGX3_S5_R1_cat.fastq.gz,fastq,4996502316.0,66258508.0,E MTAB 9727:Sample 1,0:75.41 1:0,A:1330744174;C:451622042;G:591151500;T:2622422664;N:561936,75,0,,,1330744174,451622042,591151500,2622422664,561936,ERX4665132,ERS5281173,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.29754,,0.17671,,0.9669,,0.45463,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
10067,ERR4795359,ERX4665132,ERS5281173,ERP124847,PRJEB41113,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E-MTAB-9727,Transcriptome Analysis,The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,,Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Sample 1,E MTAB 9727:Sample 1,,isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01,,,,,,,,,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,E MTAB 9727:Sample 1 s,Sample 1 s,Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 μm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 μl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5′Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at −80°C.  In brief  ERCC Spike in RNA 0.02 μL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.,Experimental Factor: replicate:lateral plate mesoderm plate 1,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,RANDOM,SINGLE,ILLUMINA,NextSeq 500,,ERP124847,NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma,ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14,SN1_AHY3WGBGX3_S5_R2_cat.fastq.gz,fastq,4999908389.0,66258508.0,E MTAB 9727:Sample 1 1,0:0 1:75.46,A:1591945117;C:933968124;G:986108614;T:1485824022;N:2062512,0,75,,,1591945117,933968124,986108614,1485824022,2062512,ERX4665132,ERS5281173,ERA3048543,University of Zurich|European Nucleotide Archive,University of Zurich|European Nucleotide Archive,1,0.81333,,0.2466,,0.83027,,0.51572,,75,,B,,usable mapping rate,illumina,nextseq,unknown,random_priming,trueseq,sc,single_cell_plate,celseq,,Switzerland,2021-04-01,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
29445,SRR27352062,SRX23028823,SRS19990706,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,xbp1 s+bhlha15 me batch C,GSM7992600,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s+bhlha15 co mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,xbp1 s+bhlha15 me batch C,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s+bhlha15 co mis expression in xbp1+atf6 double crispant,GSM7992600,GSM7992600: xbp1 s+bhlha15 me batch C; Danio rerio; RNA Seq,GSM7992600 r1,GSM7992600,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_8_S5_L001_I1_001.fastq.gz ME_E_8_S5_L001_I2_001.fastq.gz ME_E_8_S5_L001_R1_001.fastq.gz ME_E_8_S5_L001_R2_001.fastq.gz,fastq fastq fastq fastq,5181513462.0,37547199.0,GSM7992600 r1,0:10 1:10 2:28 3:90,A:956100499;C:718374108;G:793571214;T:911118244;N:83845,10,10,28,90,956100499,718374108,793571214,911118244,83845,SRX23028823,SRS19990706,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.93072,,0.1664,,0.79411,,0.55992,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29446,SRR27352063,SRX23028823,SRS19990706,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,xbp1 s+bhlha15 me batch C,GSM7992600,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s+bhlha15 co mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,xbp1 s+bhlha15 me batch C,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s+bhlha15 co mis expression in xbp1+atf6 double crispant,GSM7992600,GSM7992600: xbp1 s+bhlha15 me batch C; Danio rerio; RNA Seq,GSM7992600 r1,GSM7992600,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_8_S5_L002_I1_001.fastq.gz ME_E_8_S5_L002_I2_001.fastq.gz ME_E_8_S5_L002_R1_001.fastq.gz ME_E_8_S5_L002_R2_001.fastq.gz,fastq fastq fastq fastq,5260063890.0,38116405.0,GSM7992600 r2,0:10 1:10 2:28 3:90,A:971301150;C:729023972;G:805112224;T:924994096;N:45008,10,10,28,90,971301150,729023972,805112224,924994096,45008,SRX23028823,SRS19990706,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.92931,,0.16683,,0.79456,,0.55431,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29447,SRR27352064,SRX23028822,SRS19990705,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,bhlha15 me batch C 50pg,GSM7992599,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:bhlha15 mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,bhlha15 me batch C 50pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:bhlha15 mis expression in xbp1+atf6 double crispant,GSM7992599,GSM7992599: bhlha15 me batch C 50pg; Danio rerio; RNA Seq,GSM7992599 r1,GSM7992599,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_7_S6_L001_R2_001.fastq.gz ME_E_7_S6_L001_R1_001.fastq.gz ME_E_7_S6_L001_I2_001.fastq.gz ME_E_7_S6_L001_I1_001.fastq.gz,fastq fastq fastq fastq,6938831682.0,50281389.0,GSM7992599 r1,0:10 1:10 2:28 3:90,A:1306218371;C:953247355;G:1099874957;T:1165870220;N:114107,10,10,28,90,1306218371,953247355,1099874957,1165870220,114107,SRX23028822,SRS19990705,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.9066,,0.1671,,0.81174,,0.54662,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29448,SRR27352065,SRX23028822,SRS19990705,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,bhlha15 me batch C 50pg,GSM7992599,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:bhlha15 mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,bhlha15 me batch C 50pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:bhlha15 mis expression in xbp1+atf6 double crispant,GSM7992599,GSM7992599: bhlha15 me batch C 50pg; Danio rerio; RNA Seq,GSM7992599 r1,GSM7992599,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_7_S6_L002_R2_001.fastq.gz ME_E_7_S6_L002_R1_001.fastq.gz ME_E_7_S6_L002_I2_001.fastq.gz ME_E_7_S6_L002_I1_001.fastq.gz,fastq fastq fastq fastq,7064201370.0,51189865.0,GSM7992599 r2,0:10 1:10 2:28 3:90,A:1330402466;C:970104628;G:1119275399;T:1187243465;N:61892,10,10,28,90,1330402466,970104628,1119275399,1187243465,61892,SRX23028822,SRS19990705,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.90484,,0.16778,,0.81061,,0.523,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29449,SRR27352066,SRX23028821,SRS19990704,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,xbp1 s me batch C 70pg,GSM7992598,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,xbp1 s me batch C 70pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s mis expression in xbp1+atf6 double crispant,GSM7992598,GSM7992598: xbp1 s me batch C 70pg; Danio rerio; RNA Seq,GSM7992598 r1,GSM7992598,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_6_S7_L001_I1_001.fastq.gz ME_E_6_S7_L001_I2_001.fastq.gz ME_E_6_S7_L001_R1_001.fastq.gz ME_E_6_S7_L001_R2_001.fastq.gz,fastq fastq fastq fastq,5772411522.0,41829069.0,GSM7992598 r1,0:10 1:10 2:28 3:90,A:1071977325;C:808460271;G:918967243;T:965119818;N:91553,10,10,28,90,1071977325,808460271,918967243,965119818,91553,SRX23028821,SRS19990704,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.92142,,0.18137,,0.80886,,0.56195,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29450,SRR27352067,SRX23028821,SRS19990704,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,xbp1 s me batch C 70pg,GSM7992598,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,xbp1 s me batch C 70pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:xbp1 s mis expression in xbp1+atf6 double crispant,GSM7992598,GSM7992598: xbp1 s me batch C 70pg; Danio rerio; RNA Seq,GSM7992598 r1,GSM7992598,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_6_S7_L002_I1_001.fastq.gz ME_E_6_S7_L002_I2_001.fastq.gz ME_E_6_S7_L002_R1_001.fastq.gz ME_E_6_S7_L002_R2_001.fastq.gz,fastq fastq fastq fastq,5866797312.0,42513024.0,GSM7992598 r2,0:10 1:10 2:28 3:90,A:1090114611;C:821302663;G:933644617;T:981060240;N:50029,10,10,28,90,1090114611,821302663,933644617,981060240,50029,SRX23028821,SRS19990704,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.91937,,0.18015,,0.81201,,0.56252,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29451,SRR27352068,SRX23028820,SRS19990703,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,mCherry me batch C 120pg,GSM7992597,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,mCherry me batch C 120pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression in xbp1+atf6 double crispant,GSM7992597,GSM7992597: mCherry me batch C 120pg; Danio rerio; RNA Seq,GSM7992597 r1,GSM7992597,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_5_S8_L001_I1_001.fastq.gz ME_E_5_S8_L001_I2_001.fastq.gz ME_E_5_S8_L001_R1_001.fastq.gz ME_E_5_S8_L001_R2_001.fastq.gz,fastq fastq fastq fastq,8749401480.0,63401460.0,GSM7992597 r1,0:10 1:10 2:28 3:90,A:1660203125;C:1179357258;G:1367846490;T:1498581063;N:143464,10,10,28,90,1660203125,1179357258,1367846490,1498581063,143464,SRX23028820,SRS19990703,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.89798,,0.13736,,0.8128,,0.52474,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29452,SRR27352069,SRX23028820,SRS19990703,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,mCherry me batch C 120pg,GSM7992597,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression in xbp1+atf6 double crispant|geo loc name:missing|collection date:missing,mCherry me batch C 120pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression in xbp1+atf6 double crispant,GSM7992597,GSM7992597: mCherry me batch C 120pg; Danio rerio; RNA Seq,GSM7992597 r1,GSM7992597,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_E_5_S8_L002_I1_001.fastq.gz ME_E_5_S8_L002_I2_001.fastq.gz ME_E_5_S8_L002_R1_001.fastq.gz ME_E_5_S8_L002_R2_001.fastq.gz,fastq fastq fastq fastq,8882619504.0,64366808.0,GSM7992597 r2,0:10 1:10 2:28 3:90,A:1686216142;C:1196995709;G:1388087801;T:1521636659;N:76409,10,10,28,90,1686216142,1196995709,1388087801,1521636659,76409,SRX23028820,SRS19990703,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.89805,,0.13979,,0.81314,,0.52494,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29453,SRR27352070,SRX23028819,SRS19990702,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,creb3l2 N me 40pg,GSM7992596,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 N mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,creb3l2 N me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 N mis expression in xbp1 crispant,GSM7992596,GSM7992596: creb3l2 N me 40pg; Danio rerio; RNA Seq,GSM7992596 r1,GSM7992596,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_6_S6_L001_R2_001.fastq.gz ME_D_6_S6_L001_R1_001.fastq.gz ME_D_6_S6_L001_I1_001.fastq.gz,fastq fastq fastq,53307532026.0,423075651.0,GSM7992596 r1,0:8 1:28 2:90,A:10926383189;C:7941787834;G:8886027055;T:10318943731;N:3666781,8,28,90,,10926383189,7941787834,8886027055,10318943731,3666781,SRX23028819,SRS19990702,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.91179,,0.15023,,0.80359,,0.54471,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29454,SRR27352071,SRX23028819,SRS19990702,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,creb3l2 N me 40pg,GSM7992596,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 N mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,creb3l2 N me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 N mis expression in xbp1 crispant,GSM7992596,GSM7992596: creb3l2 N me 40pg; Danio rerio; RNA Seq,GSM7992596 r1,GSM7992596,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_6_S7_L004_R2_001.fastq.gz ME_D_6_S7_L004_R1_001.fastq.gz ME_D_6_S7_L004_I1_001.fastq.gz,fastq fastq fastq,21499800210.0,102380001.0,GSM7992596 r2,0:8 1:101 2:101,A:5731045724;C:3931487777;G:4014771792;T:7002424207;N:1030702,8,101,101,,5731045724,3931487777,4014771792,7002424207,1030702,SRX23028819,SRS19990702,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",2,0.0,0.91139,0.0,0.15189,1.0,0.80702,,0.54085,101,101,T,B,mate1 technical by mapping diff,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29455,SRR27352072,SRX23028818,SRS19990701,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,creb3l1 N me 40pg,GSM7992595,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l1 N mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,creb3l1 N me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l1 N mis expression in xbp1 crispant,GSM7992595,GSM7992595: creb3l1 N me 40pg; Danio rerio; RNA Seq,GSM7992595 r1,GSM7992595,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_5_S5_L001_I1_001.fastq.gz ME_D_5_S5_L001_R1_001.fastq.gz ME_D_5_S5_L001_R2_001.fastq.gz,fastq fastq fastq,39710838132.0,315165382.0,GSM7992595 r1,0:8 1:28 2:90,A:8134239938;C:5939509853;G:6667896902;T:7620537309;N:2700378,8,28,90,,8134239938,5939509853,6667896902,7620537309,2700378,SRX23028818,SRS19990701,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.91062,,0.15355,,0.80432,,0.56447,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29456,SRR27352073,SRX23028818,SRS19990701,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,creb3l1 N me 40pg,GSM7992595,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l1 N mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,creb3l1 N me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l1 N mis expression in xbp1 crispant,GSM7992595,GSM7992595: creb3l1 N me 40pg; Danio rerio; RNA Seq,GSM7992595 r1,GSM7992595,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_5_S6_L004_I1_001.fastq.gz ME_D_5_S6_L004_R1_001.fastq.gz ME_D_5_S6_L004_R2_001.fastq.gz,fastq fastq fastq,24091335030.0,114720643.0,GSM7992595 r2,0:8 1:101 2:101,A:6381365861;C:4422323917;G:4533780207;T:7834947754;N:1152147,8,101,101,,6381365861,4422323917,4533780207,7834947754,1152147,SRX23028818,SRS19990701,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",2,0.0,0.90836,0.0,0.1531,1.0,0.80718,,0.56329,101,101,T,B,mate1 technical by mapping diff,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29473,SRR27352090,SRX23028809,SRS19990692,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,atf6 N me 40pg,GSM7992594,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:atf6 N mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,atf6 N me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:atf6 N mis expression in xbp1 crispant,GSM7992594,GSM7992594: atf6 N me 40pg; Danio rerio; RNA Seq,GSM7992594 r1,GSM7992594,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_4_S7_L001_R2_001.fastq.gz ME_D_4_S7_L001_R1_001.fastq.gz ME_D_4_S7_L001_I1_001.fastq.gz,fastq fastq fastq,34201406232.0,271439732.0,GSM7992594 r1,0:8 1:28 2:90,A:6970137729;C:5129294511;G:5749174804;T:6578660562;N:2308274,8,28,90,,6970137729,5129294511,5749174804,6578660562,2308274,SRX23028809,SRS19990692,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.91553,,0.16107,,0.79835,,0.5523,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29474,SRR27352091,SRX23028809,SRS19990692,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,atf6 N me 40pg,GSM7992594,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:atf6 N mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,atf6 N me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:atf6 N mis expression in xbp1 crispant,GSM7992594,GSM7992594: atf6 N me 40pg; Danio rerio; RNA Seq,GSM7992594 r1,GSM7992594,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_4_S8_L004_R2_001.fastq.gz ME_D_4_S8_L004_R1_001.fastq.gz ME_D_4_S8_L004_I1_001.fastq.gz,fastq fastq fastq,22385290830.0,106596623.0,GSM7992594 r2,0:8 1:101 2:101,A:5920329288;C:4138677002;G:4227668821;T:7244770032;N:1072703,8,101,101,,5920329288,4138677002,4227668821,7244770032,1072703,SRX23028809,SRS19990692,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",2,0.0,0.91524,0.0,0.16081,1.0,0.80113,,0.55582,101,101,T,B,mate1 technical by mapping diff,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29475,SRR27352092,SRX23028808,SRS19990691,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,mCherry me 40pg,GSM7992593,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:NLS mCherry mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,mCherry me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:NLS mCherry mis expression in xbp1 crispant,GSM7992593,GSM7992593: mCherry me 40pg; Danio rerio; RNA Seq,GSM7992593 r1,GSM7992593,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_3_S4_L001_R2_001.fastq.gz ME_D_3_S4_L001_R1_001.fastq.gz ME_D_3_S4_L001_I1_001.fastq.gz,fastq fastq fastq,31946333832.0,253542332.0,GSM7992593 r1,0:8 1:28 2:90,A:6560614856;C:4781373893;G:5380587448;T:6094065607;N:2168076,8,28,90,,6560614856,4781373893,5380587448,6094065607,2168076,SRX23028808,SRS19990691,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.90476,,0.15614,,0.81391,,0.56731,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29476,SRR27352093,SRX23028808,SRS19990691,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,mCherry me 40pg,GSM7992593,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:NLS mCherry mis expression in xbp1 crispant|geo loc name:missing|collection date:missing,mCherry me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:NLS mCherry mis expression in xbp1 crispant,GSM7992593,GSM7992593: mCherry me 40pg; Danio rerio; RNA Seq,GSM7992593 r1,GSM7992593,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_3_S5_L004_I1_001.fastq.gz ME_D_3_S5_L004_R1_001.fastq.gz ME_D_3_S5_L004_R2_001.fastq.gz,fastq fastq fastq,23152132290.0,110248249.0,GSM7992593 r2,0:8 1:101 2:101,A:6128844846;C:4261101648;G:4373973829;T:7505119299;N:1106676,8,101,101,,6128844846,4261101648,4373973829,7505119299,1106676,SRX23028808,SRS19990691,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",2,0.0,0.90407,0.0,0.15893,1.0,0.81734,,0.56376,101,101,T,B,mate1 technical by mapping diff,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29477,SRR27352094,SRX23028807,SRS19990690,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,creb3l2 full me 40pg,GSM7992592,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 full length mis exprerssion|geo loc name:missing|collection date:missing,creb3l2 full me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 full length mis exprerssion,GSM7992592,GSM7992592: creb3l2 full me 40pg; Danio rerio; RNA Seq,GSM7992592 r1,GSM7992592,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_2_S13_L001_I1_001.fastq.gz ME_D_2_S13_L001_R1_001.fastq.gz ME_D_2_S13_L001_R2_001.fastq.gz,fastq fastq fastq,15222166848.0,120810848.0,GSM7992592 r1,0:8 1:28 2:90,A:3076061131;C:2328534823;G:2595273328;T:2872069911;N:1037127,8,28,90,,3076061131,2328534823,2595273328,2872069911,1037127,SRX23028807,SRS19990690,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.91719,,0.16191,,0.80837,,0.55344,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29478,SRR27352095,SRX23028807,SRS19990690,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,creb3l2 full me 40pg,GSM7992592,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 full length mis exprerssion|geo loc name:missing|collection date:missing,creb3l2 full me 40pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:creb3l2 full length mis exprerssion,GSM7992592,GSM7992592: creb3l2 full me 40pg; Danio rerio; RNA Seq,GSM7992592 r1,GSM7992592,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_2_S14_L004_I1_001.fastq.gz ME_D_2_S14_L004_R1_001.fastq.gz ME_D_2_S14_L004_R2_001.fastq.gz,fastq fastq fastq,20633376540.0,98254174.0,GSM7992592 r2,0:8 1:101 2:101,A:5431963590;C:3898811025;G:3951937782;T:6563643256;N:987495,8,101,101,,5431963590,3898811025,3951937782,6563643256,987495,SRX23028807,SRS19990690,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",2,0.0,0.91717,0.0,0.16142,1.0,0.81063,,0.52718,101,101,T,B,mate1 technical by mapping diff,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29479,SRR27352096,SRX23028806,SRS19990689,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,mCherry me 125pg,GSM7992591,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression|geo loc name:missing|collection date:missing,mCherry me 125pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression,GSM7992591,GSM7992591: mCherry me 125pg; Danio rerio; RNA Seq,GSM7992591 r1,GSM7992591,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_1_S11_L001_R2_001.fastq.gz ME_D_1_S11_L001_R1_001.fastq.gz ME_D_1_S11_L001_I1_001.fastq.gz,fastq fastq fastq,7592631102.0,60258977.0,GSM7992591 r1,0:8 1:28 2:90,A:1549191430;C:1138023380;G:1260358863;T:1475225062;N:509195,8,28,90,,1549191430,1138023380,1260358863,1475225062,509195,SRX23028806,SRS19990689,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",1,0.91893,,0.15048,,0.80517,,0.54261,,90,,B,,usable mapping rate,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
29480,SRR27352097,SRX23028806,SRS19990689,SRP480147,PRJNA1057153,Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion,GSE252032,Transcriptome Analysis,During differentiation  cells become structurally and functionally specialized  but comprehensive views of the underlying remodeling processes are elusive. Here  we leverage scRNA seq developmental trajectories to reconstruct differentiation  using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First  we present an approach MIMIR to integrate expression and functional similarities for gene module identification  revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second  we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss  and gain of function embryos  we found that the UPR transcription factors creb3l1  creb3l2  and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program  while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1  atf6  creb3l1  and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs  we performed scRNA seq of UPR TF loss of function embryos at 12 hpf  when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double  triple  and quadruple loss of function embryos. Tyrosinase tyr crispants  which do not exhibit developmental defects  were generated as the control. Using this loss of function dataset  we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next  we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos  we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf  when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable  and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1  creb3l2  and atf6  and atf6 can be activated by xbp1  we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control  the activated forms of xbp1 xbp1 s  creb3l1 creb3l1 N  creb3l2 creb3l2 N  and atf6 atf6 N  as well as the non activated  full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.,,pubmed:38234833;pubmed:39591963,,mCherry me 125pg,GSM7992591,,source name:Whole embryo|tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression|geo loc name:missing|collection date:missing,mCherry me 125pg,"The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the ""cellranger count"" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8  the reference transcriptome was modified: the mis expressed  codon optimized  xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the ""cellranger count"" command. barcodes.tsv.gz  features.tsv.gz  and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.",Whole embryo,1 cell stage embryos were either injected with Cas9 and gRNA  injected with mRNA  or uninjected according to experimental conditions.,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.,tissue:Whole embryo|strain:TL/AB|developmental stage:8 hpf|genotype:nls mCherry mis expression,GSM7992591,GSM7992591: mCherry me 125pg; Danio rerio; RNA Seq,GSM7992591 r1,GSM7992591,1,At desired developmental stages 8hpf or 12hpf  approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry  embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl  1.8 mM KCl  1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5  110 mM NaCl  3.5 mM KCl  2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco  11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more  with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry  several modifications were made to the dissociation protocol described above. Briefly  post being transferred to 2ml Eppendorf tubes  embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma  P5380]  125 U/ml DNaseI  2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS  0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging  resuspending  and filtering steps were then performed as in the previous protocol  but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP480147,,loader:fastq load.py,ME_D_1_S12_L004_I1_001.fastq.gz ME_D_1_S12_L004_R1_001.fastq.gz ME_D_1_S12_L004_R2_001.fastq.gz,fastq fastq fastq,22469033160.0,106995396.0,GSM7992591 r2,0:8 1:101 2:101,A:5978154538;C:4132913966;G:4202172487;T:7298755208;N:1073793,8,101,101,,5978154538,4132913966,4202172487,7298755208,1073793,SRX23028806,SRS19990689,SRA1775820,"Schier Lab, Harvard University","Schier Lab, Harvard University",2,0.0,0.91624,0.0,0.15006,1.0,0.80643,,0.54121,101,101,T,B,mate1 technical by mapping diff,illumina,novaseq_era,full_length,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2023-12-26,Gastrula,Embryo,Whole Organism,All anatomical structures
32134,SRR29020075,SRX24545646,SRS21290023,SRP507567,PRJNA1111448,rbm24a is an organizer component of germ plasm to determine germ cell fate,GSE267406,Transcriptome Analysis,The  formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants  but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs  which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows  where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs  resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm  highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: We performed single cell RNA seq scRNA seq for shield stage embryos to compare gene expression between Mrbm24a and sibling embryos. Another purpose of this omic analysis is to examine possible cell populations that can be affected by the loss of maternal Rbm24a.,,,,Zebrafish  scRNAseq  shield sibling 2,GSM8265296,,source name:whole embryo|tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:wild type|geo loc name:missing|collection date:missing,Zebrafish  scRNAseq  shield sibling 2,To remove any remaining biochemical reagents and primers from the post GEM reaction mixture  silane magnetic beads were employed. The full length  barcoded cDNAs were subsequently amplified by PCR to generate sufficient mass for library construction. During GEM incubation  the molecules had R1 Read 1 primer sequence added to them. P5  P7  a sample index  and R2 Read 2 primer sequence were added during library construction through processes such as End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries contained the P5 and P7 primers required for Illumina bridge amplification. The Single Cell three prime Protocol generated Illumina ready sequencing libraries. Each Single Cell three prime Library consisted of standard Illumina paired end constructs that began and ended with P5 and P7. The Single Cell three prime 16 bp 10x Barcode and 10 bp UMI were encoded in Read 1  while Read 2 was utilized to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 corresponded to the standard Illumina® sequencing primer sites used in paired end sequencing. Assembly: GRCz10 Supplementary files format and content: Tab separated values files and matrix files,whole embryo,,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C,tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:wild type,GSM8265296,GSM8265296: Zebrafish  scRNAseq  shield sibling 2; Danio rerio; RNA Seq,GSM8265296 r1,GSM8265296,1,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP507567,,,J23090498_R2_001.fastq.gz J23090498_R1_001.fastq.gz,fastq fastq,106757315400.0,355857718.0,GSM8265296 r1,0:150 1:150,A:33082219289;C:20599674613;G:20857073591;T:32217553311;N:794596,150,150,,,33082219289,20599674613,20857073591,32217553311,794596,SRX24545646,SRS21290023,SRA1866413,"shao ming lab, school of life sciences, shandong university","shao ming lab, school of life sciences, shandong university",2,0.2631,0.91686,0.0934,0.16341,0.98742,0.78798,0.70164,0.53109,150,150,T,B,mate1 technical by mapping diff,illumina,nextseq,3prime,random_priming,unknown,sc,single_cell_droplet,10x,,China,2024-05-14,Gastrula,Embryo,Whole Organism,All anatomical structures
32135,SRR29020076,SRX24545645,SRS21290022,SRP507567,PRJNA1111448,rbm24a is an organizer component of germ plasm to determine germ cell fate,GSE267406,Transcriptome Analysis,The  formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants  but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs  which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows  where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs  resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm  highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: We performed single cell RNA seq scRNA seq for shield stage embryos to compare gene expression between Mrbm24a and sibling embryos. Another purpose of this omic analysis is to examine possible cell populations that can be affected by the loss of maternal Rbm24a.,,,,Zebrafish  scRNAseq  shield sibling 1,GSM8265295,,source name:whole embryo|tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:wild type|geo loc name:missing|collection date:missing,Zebrafish  scRNAseq  shield sibling 1,To remove any remaining biochemical reagents and primers from the post GEM reaction mixture  silane magnetic beads were employed. The full length  barcoded cDNAs were subsequently amplified by PCR to generate sufficient mass for library construction. During GEM incubation  the molecules had R1 Read 1 primer sequence added to them. P5  P7  a sample index  and R2 Read 2 primer sequence were added during library construction through processes such as End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries contained the P5 and P7 primers required for Illumina bridge amplification. The Single Cell three prime Protocol generated Illumina ready sequencing libraries. Each Single Cell three prime Library consisted of standard Illumina paired end constructs that began and ended with P5 and P7. The Single Cell three prime 16 bp 10x Barcode and 10 bp UMI were encoded in Read 1  while Read 2 was utilized to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 corresponded to the standard Illumina® sequencing primer sites used in paired end sequencing. Assembly: GRCz10 Supplementary files format and content: Tab separated values files and matrix files,whole embryo,,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C,tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:wild type,GSM8265295,GSM8265295: Zebrafish  scRNAseq  shield sibling 1; Danio rerio; RNA Seq,GSM8265295 r1,GSM8265295,1,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP507567,,,J23090497_R1_001.fastq.gz J23090497_R2_001.fastq.gz,fastq fastq,106697824200.0,355659414.0,GSM8265295 r1,0:150 1:150,A:33526661561;C:19978357135;G:20431377225;T:32760687892;N:740387,150,150,,,33526661561,19978357135,20431377225,32760687892,740387,SRX24545645,SRS21290022,SRA1866413,"shao ming lab, school of life sciences, shandong university","shao ming lab, school of life sciences, shandong university",2,0.16292,0.91415,0.04208,0.15118,0.98859,0.78914,0.58909,0.51597,150,150,T,B,mate1 technical by mapping diff,illumina,nextseq,3prime,random_priming,unknown,sc,single_cell_droplet,10x,,China,2024-05-14,Gastrula,Embryo,Whole Organism,All anatomical structures
32136,SRR29020077,SRX24545644,SRS21290021,SRP507567,PRJNA1111448,rbm24a is an organizer component of germ plasm to determine germ cell fate,GSE267406,Transcriptome Analysis,The  formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants  but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs  which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows  where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs  resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm  highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: We performed single cell RNA seq scRNA seq for shield stage embryos to compare gene expression between Mrbm24a and sibling embryos. Another purpose of this omic analysis is to examine possible cell populations that can be affected by the loss of maternal Rbm24a.,,,,Zebrafish  scRNAseq  shield Mrbm24a 2,GSM8265294,,source name:whole embryo|tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:Mrbm24a|geo loc name:missing|collection date:missing,Zebrafish  scRNAseq  shield Mrbm24a 2,To remove any remaining biochemical reagents and primers from the post GEM reaction mixture  silane magnetic beads were employed. The full length  barcoded cDNAs were subsequently amplified by PCR to generate sufficient mass for library construction. During GEM incubation  the molecules had R1 Read 1 primer sequence added to them. P5  P7  a sample index  and R2 Read 2 primer sequence were added during library construction through processes such as End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries contained the P5 and P7 primers required for Illumina bridge amplification. The Single Cell three prime Protocol generated Illumina ready sequencing libraries. Each Single Cell three prime Library consisted of standard Illumina paired end constructs that began and ended with P5 and P7. The Single Cell three prime 16 bp 10x Barcode and 10 bp UMI were encoded in Read 1  while Read 2 was utilized to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 corresponded to the standard Illumina® sequencing primer sites used in paired end sequencing. Assembly: GRCz10 Supplementary files format and content: Tab separated values files and matrix files,whole embryo,,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C,tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:Mrbm24a,GSM8265294,GSM8265294: Zebrafish  scRNAseq  shield Mrbm24a 2; Danio rerio; RNA Seq,GSM8265294 r1,GSM8265294,1,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP507567,,,J23090496_R2_001.fastq.gz J23090496_R1_001.fastq.gz,fastq fastq,107289285600.0,357630952.0,GSM8265294 r1,0:150 1:150,A:33671128260;C:20203779331;G:20583824987;T:32829771822;N:781200,150,150,,,33671128260,20203779331,20583824987,32829771822,781200,SRX24545644,SRS21290021,SRA1866413,"shao ming lab, school of life sciences, shandong university","shao ming lab, school of life sciences, shandong university",2,0.17098,0.91193,0.0312,0.14762,0.98772,0.789,0.58127,0.51217,150,150,T,B,mate1 technical by mapping diff,illumina,nextseq,3prime,random_priming,unknown,sc,single_cell_droplet,10x,,China,2024-05-14,Gastrula,Embryo,Whole Organism,All anatomical structures
32137,SRR29020078,SRX24545643,SRS21290020,SRP507567,PRJNA1111448,rbm24a is an organizer component of germ plasm to determine germ cell fate,GSE267406,Transcriptome Analysis,The  formation of germ cells is a critical issue for the continuation of species. A large group of animals follow a preformation strategy to generate their primordial germ cells PGCs. They produce a set of localized maternal mRNAs and proteins to form phase separated germ plasm that functions as the PGC determinants  but the mechanisms underlying the assembly of germ plasm is poorly understood. This study identifies Rbm24a as an enssential localized germ plasm protein component that controls the formation of large and functional germ plasm granules. Rbm24a is complexed with Buc and interacts with germ plasm mRNAs  which determines the specific grasp of germ plasm mRNAs into the phase separated aggregates. Rbm24a absent granules fail to undergo kinesin dependent transport towards the cleavage furrows  where small particles fuse into large ones. The loss of maternal rbm24a causes the complete degradation of germ plasm components and the disappearance of PGCs  resulting in totally sterile animals. Our work establishes that Rbm24 is a critical nucleating organizer component of germ plasm  highlighting an emerging common mechanism to read and recruit RNA component into phase separated condensates. Overall design: We performed single cell RNA seq scRNA seq for shield stage embryos to compare gene expression between Mrbm24a and sibling embryos. Another purpose of this omic analysis is to examine possible cell populations that can be affected by the loss of maternal Rbm24a.,,,,Zebrafish  scRNAseq  shield Mrbm24a 1,GSM8265293,,source name:whole embryo|tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:Mrbm24a|geo loc name:missing|collection date:missing,Zebrafish  scRNAseq  shield Mrbm24a 1,To remove any remaining biochemical reagents and primers from the post GEM reaction mixture  silane magnetic beads were employed. The full length  barcoded cDNAs were subsequently amplified by PCR to generate sufficient mass for library construction. During GEM incubation  the molecules had R1 Read 1 primer sequence added to them. P5  P7  a sample index  and R2 Read 2 primer sequence were added during library construction through processes such as End Repair  A tailing  Adaptor Ligation  and PCR. The final libraries contained the P5 and P7 primers required for Illumina bridge amplification. The Single Cell three prime Protocol generated Illumina ready sequencing libraries. Each Single Cell three prime Library consisted of standard Illumina paired end constructs that began and ended with P5 and P7. The Single Cell three prime 16 bp 10x Barcode and 10 bp UMI were encoded in Read 1  while Read 2 was utilized to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 corresponded to the standard Illumina® sequencing primer sites used in paired end sequencing. Assembly: GRCz10 Supplementary files format and content: Tab separated values files and matrix files,whole embryo,,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,Zebrafish embryos were maintained in 1/3x Ringer's at 28.5C,tissue:whole embryo|cell line:shield stage|cell type:embryonic cell|genotype:Mrbm24a,GSM8265293,GSM8265293: Zebrafish  scRNAseq  shield Mrbm24a 1; Danio rerio; RNA Seq,GSM8265293 r1,GSM8265293,1,For the preparation of single cell suspensions  a total of thirty three mutant embryos of Mrbm24a or wild type embryos at the shield stage were collected. These cellular suspensions were then loaded onto a 10X Genomics GemCode Single cell instrument  which generates single cell Gel Bead In EMulsion GEMs. Libraries were generated and sequenced from the cDNAs using Chromium Next GEM Single Cell three prime Reagent Kits v3.1. Upon dissolution of the Gel Bead within a GEM  primers containing an Illumina® R1 sequence Read 1 sequencing primer  a 16 nt 10x Barcode  a 10 nt Unique Molecular Identifier UMI  and a poly dT primer sequence were released. These primers were mixed with the cell lysate and Master Mix. Full length  barcoded cDNAs were then reverse transcribed from poly adenylated mRNA,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP507567,,,J23090495_R1_001.fastq.gz J23090495_R2_001.fastq.gz,fastq fastq,98193373500.0,327311245.0,GSM8265293 r1,0:150 1:150,A:27502195548;C:18458481431;G:18979601074;T:33252654514;N:440933,150,150,,,27502195548,18458481431,18979601074,33252654514,440933,SRX24545643,SRS21290020,SRA1866413,"shao ming lab, school of life sciences, shandong university","shao ming lab, school of life sciences, shandong university",2,0.00149,0.9183,0.00037,0.14737,0.99947,0.79125,0.51851,0.50318,150,150,T,B,mate1 technical by mapping diff,illumina,nextseq,3prime,random_priming,unknown,sc,single_cell_droplet,10x,,China,2024-05-14,Gastrula,Embryo,Whole Organism,All anatomical structures
33864,SRR30779425,SRX26181412,SRS22725400,SRP534298,PRJNA1164307,Time resolved single cell Multiomic zebrafish atlas,PRJNA1164307,Other,During development  dynamic interplay between transcription factors  chromatin  and genes  termed gene regulatory network GRN  shapes the cell fate determination along the developmental trajectory. Recent advances in joint measurement of chromatin accessibility and gene expression enabled the genome wide identification of regulatory relationships. Here  we assess the dynamics of the gene regulatory network in zebrafish development using joint single cell ATAC and single cell RNA sequencing. We discovered some key regulatory modules that exhibit cell type and time dependent activity  suggesting that the role of transcription factors vary over cell type and timepoints. With time resolved GRNs combined with linear modeling framework  we performed a systematic in silico knock out simulation using CellOracle. This in silico knock out simulation revealed that the role of transcription factors is shared between mesodermal and neuro ectodermal lineages in the early timepoints  but later commit significantly to either lineages. Together  we provide a dataset and a framework to systematically dissect the role of transcription factors during the zebrafish embryonic development.,,,,TDR126,TDR126 10hpf EKW NA none 10xmultiome,,strain:EKW|dev stage:Bud stage 10 hpf|collection date:2023 11 22|geo loc name:USA: San Francisco|sex:N/A|tissue:organism|BioSampleModel:Model organism or animal,,,,,,,,,TDR126 10hpf RNA,TDR126 10hpf RNA EKW NA none 10xmultiome,TDR126 10hpf RNA EKW NA none 10xmultiome,10x multiome nuc seq,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP534298,,,TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L001_I1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L001_I2_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L001_R1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L001_R2_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L002_I1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L002_I2_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L002_R1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L002_R2_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L003_I1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L003_I2_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L003_R1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L003_R2_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L004_I1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L004_I2_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L004_R1_001.fastq.gz TDR126_10hpf_RNA_EKW_NA_none_10xmultiome_S4_L004_R2_001.fastq.gz,fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq,,,TDR126 10hpf RNA EKW NA none 10xmultiome S4 L001 I1 001.fastq.gz,,,,,,,,,,,,SRX26181412,,SRA1977819,Chan Zuckerberg Biohub San Francisco|Computational Biology,Chan Zuckerberg Biohub San Francisco,,,,,,,,,,,,,,,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United States,2024-09-23,Gastrula,Embryo,Undetermined,Embryo Imprecise
72588,SRR22797023,SRX18756814,SRS16192272,SRP413556,PRJNA913420,Nodal coordinates the anterior posterior patterning of germ layers and induces head formation in zebrafish explants.,GSE221220,Other,Purpose: To investingate cell types in wild type zebrafish explants. Methods:  Wild type explants injected with phenol red were harvested at xxxhpf. Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000075 according to the manufacturer's protocol for 10000 cells recovery. Results: A total of 9 968 single cell transcriptomes were collected post stringent quality control measures. Conclusions: 6 Cell types  were identified  wild type explants mainly contains the anterior neural ectoderm and epidermis at 10 hpf. Overall design: Wild type zebrafish explants were harvested at 10hpf for scRNA seq.,,pubmed:37729057,,zebrafish wild type explant 10hpf,GSM6856370,,source name:zebrafish cells|strain:AB|tissue:embryonic cells|age:10hpf|genotype:wild type,zebrafish wild type explant 10hpf,Illumina sequencing reads were aligned to the zebrafish mRNA reference genome GRCz11 using the 10x Genomics CellRanger pipeline version 3.0.2 with default parameters. Assembly: GRCz11 Supplementary files format and content: tar compressed  files included filtered gene bc matrices post running CellRanger pipeline,zebrafish cells,phenol red was injected to one cell of embryonic animal pole at xxx cell stage. All embryos were incubated in 0.3x Danieau buffer until 1k stage  then  were transferred to Dulbecco's Modified Eagle Medium. Animal pole explants corresponding roughly to half of the blastula were incubated to 10hpfcorresponding to embryonic developmental stage separately.,Libraries were prepared using Chromium Controller and Chromium Single Cell 3’Library & Gel Bead Kit v3 10x Genomics  PN 1000075 according to the manufacturer’s protocol for 10000 cells recovery.,explants were cultured in a Petri dish coated with 1.5% agarose filled with Dulbecco's Modified Eagle Medium,strain:AB|tissue:embryonic cells|age:10hpf|genotype:wild type,GSM6856370,GSM6856370: zebrafish wild type explant 10hpf; Danio rerio; RNA Seq,GSM6856370 r1,GSM6856370,1,Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000075 according to the manufacturer's protocol for 10000 cells recovery.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP413556,,loader:fastq load.py,WT_10hpf_S1_L001_R1_001.fastq.gz WT_10hpf_S1_L001_R2_001.fastq.gz,fastq fastq,144284560800.0,480948536.0,GSM6856370 r1,0:150 1:150,A:61459567540;C:25433070866;G:24459703281;T:32928214337;N:4004776,150,150,,,61459567540,25433070866,24459703281,32928214337,4004776,SRX18756814,SRS16192272,SRA1561216,"Institute of genetics, Zhejiang University","Institute of genetics, Zhejiang University",2,0.0,0.92423,0.0,0.16334,1.0,0.78228,,0.51889,150,150,T,B,mate1 technical by mapping diff,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2022-12-17,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
72834,SRR23210473,SRX19158410,SRS16570831,SRP418915,PRJNA927004,Differential Nodal level promotes mesendoderm cell fate segregation mediated by chromatin organization,GSE223636,Other,Purpose: To investigate the mechanism of prechordal plate and anterior endoderm separation . Methods:  Nodal injected explants injected with 10pg ndr2 mRNA constructed from lft1 mutants and ndr1 morphants were harvested at xxxhpf. Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000075 according to the manufacturer's protocol for 10000 cells recovery. For single cell multiomics  zebrafish embryos at 6 hpf were harvested.  Libraries were prepared using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x Genomics  4 rxns PN 1000285 according to the manufacturer's protocol for 10000 cells recovery. Results: A total of 10 614 single cell transcriptomes and 4 335 multiomics were collected post stringent quality control measures. Conclusions: A slight bias in Nodal signaling promotes a differential chromatin structure between prechordal plate and endoderm  which drives a differential expression of those key regulators  such as gsc and ripply1 in these two cell lineages  and further regulates mesendoderm cell fate separation. Overall design: zebrafish Nodal explants constructed from lft1 mutants and ndr1 morphants were harvested at 6hpf for scRNA seq. Zebrafish embryos were harvested at 6hpf for single cell multiomics.,,,,zebrafish Nodal explants constructed from ndr1 morphants,GSM6969676,,source name:zebrafish cells|strain:AB|tissue:embryonic cells|age:6hpf,zebrafish Nodal explants constructed from ndr1 morphants,Illumina sequencing reads were aligned to the zebrafish mRNA reference genome GRCz11 using the 10x Genomics CellRanger pipeline version 6.1.2 and cellranger arc version 2.0.0 with default parameters. Assembly: GRCz11 Supplementary files format and content: tar archivr or  gzip compressed  files included filtered gene bc matrices and ATAC fragments  post running CellRanger or cellranger arc pipeline.,zebrafish cells,10pg of ndr2 mRNA was injected to one cell of embryonic animal pole at xxx cell stage. All embryos were incubated in 0.3x Danieau buffer until 1k stage  then  were transferred to Dulbecco's Modified Eagle Medium. Animal pole explants corresponding roughly to half of the blastula were incubated to 6hpf corresponding to embryonic developmental stage.,Libraries were prepared using Chromium Controller and Chromium Single Cell 3’Library & Gel Bead Kit v3 10x Genomics  PN 1000075 and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x Genomics  4 rxns PN 1000285 according to the manufacturer’s protocol for 10000 cells recovery.,Explants were cultured in a Petri dish coated with 1.5% agarose filled with Dulbecco's Modified Eagle Medium,strain:AB|tissue:embryonic cells|age:6hpf,GSM6969676,GSM6969676: zebrafish Nodal explants constructed from ndr1 morphants; Danio rerio; RNA Seq,GSM6969676 r1,GSM6969676,1,Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000075 and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x Genomics  4 rxns PN 1000285 according to the manufacturer's protocol for 10000 cells recovery.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP418915,,,ndr1Mo_cyc_6hpf_S5_L002_R1_001.fastq.gz ndr1Mo_cyc_6hpf_S5_L002_R2_001.fastq.gz,fastq fastq,82246861872.0,464671536.0,GSM6969676 r1,0:26 1:151,A:23306781803;C:17200141798;G:18609129782;T:23129217367;N:1591122,26,151,,,23306781803,17200141798,18609129782,23129217367,1591122,SRX19158410,SRS16570831,SRA1581154,"Institute of genetics, Zhejiang University","Institute of genetics, Zhejiang University",2,0.00753,0.93187,0.00244,0.09764,0.9867,0.79813,0.42974,0.53736,26,151,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2023-01-24,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
72835,SRR23210474,SRX19158409,SRS16570830,SRP418915,PRJNA927004,Differential Nodal level promotes mesendoderm cell fate segregation mediated by chromatin organization,GSE223636,Other,Purpose: To investigate the mechanism of prechordal plate and anterior endoderm separation . Methods:  Nodal injected explants injected with 10pg ndr2 mRNA constructed from lft1 mutants and ndr1 morphants were harvested at xxxhpf. Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000075 according to the manufacturer's protocol for 10000 cells recovery. For single cell multiomics  zebrafish embryos at 6 hpf were harvested.  Libraries were prepared using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x Genomics  4 rxns PN 1000285 according to the manufacturer's protocol for 10000 cells recovery. Results: A total of 10 614 single cell transcriptomes and 4 335 multiomics were collected post stringent quality control measures. Conclusions: A slight bias in Nodal signaling promotes a differential chromatin structure between prechordal plate and endoderm  which drives a differential expression of those key regulators  such as gsc and ripply1 in these two cell lineages  and further regulates mesendoderm cell fate separation. Overall design: zebrafish Nodal explants constructed from lft1 mutants and ndr1 morphants were harvested at 6hpf for scRNA seq. Zebrafish embryos were harvested at 6hpf for single cell multiomics.,,,,zebrafish Nodal explants constructed from lft1 mutants,GSM6969675,,source name:zebrafish cells|strain:lft1 mutant|tissue:embryonic cells|age:6hpf,zebrafish Nodal explants constructed from lft1 mutants,Illumina sequencing reads were aligned to the zebrafish mRNA reference genome GRCz11 using the 10x Genomics CellRanger pipeline version 6.1.2 and cellranger arc version 2.0.0 with default parameters. Assembly: GRCz11 Supplementary files format and content: tar archivr or  gzip compressed  files included filtered gene bc matrices and ATAC fragments  post running CellRanger or cellranger arc pipeline.,zebrafish cells,10pg of ndr2 mRNA was injected to one cell of embryonic animal pole at xxx cell stage. All embryos were incubated in 0.3x Danieau buffer until 1k stage  then  were transferred to Dulbecco's Modified Eagle Medium. Animal pole explants corresponding roughly to half of the blastula were incubated to 6hpf corresponding to embryonic developmental stage.,Libraries were prepared using Chromium Controller and Chromium Single Cell 3’Library & Gel Bead Kit v3 10x Genomics  PN 1000075 and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x Genomics  4 rxns PN 1000285 according to the manufacturer’s protocol for 10000 cells recovery.,Explants were cultured in a Petri dish coated with 1.5% agarose filled with Dulbecco's Modified Eagle Medium,strain:lft1 mutant|tissue:embryonic cells|age:6hpf,GSM6969675,GSM6969675: zebrafish Nodal explants constructed from lft1 mutants; Danio rerio; RNA Seq,GSM6969675 r1,GSM6969675,1,Libraries were prepared using Chromium Controller and Chromium Single Cell three primeLibrary & Gel Bead Kit v3 10x Genomics  PN 1000075 and Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle 10x Genomics  4 rxns PN 1000285 according to the manufacturer's protocol for 10000 cells recovery.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP418915,,,lft1Mu_cyc_6hpf_S1_L001_R1_001.fastq.gz lft1Mu_cyc_6hpf_S1_L001_R2_001.fastq.gz,fastq fastq,89053334922.0,503126186.0,GSM6969675 r1,0:26 1:151,A:26667371583;C:17898813431;G:19518773309;T:24966420492;N:1956107,26,151,,,26667371583,17898813431,19518773309,24966420492,1956107,SRX19158409,SRS16570830,SRA1581154,"Institute of genetics, Zhejiang University","Institute of genetics, Zhejiang University",2,0.01205,0.89743,0.00297,0.09338,0.98082,0.80961,0.48847,0.58599,26,151,T,B,sc-like readlen,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,China,2023-01-24,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
74105,SRR23380800,SRX19321399,SRS16719697,SRP422528,PRJNA934545,A single cell map of maternal and zygotic mRNA dynamics during cell type specification in zebrafish embryos [scSLAM seq],GSE224918,Other,During early embryogenesis  embryos undergo a massive degradation of maternally inherited mRNAs and produce new zygotic transcripts. This maternal to zygotic transition requires a tight interplay of mRNA transcription and degradation  but distinguishing their unique contributions remains a challenge. Here  we dissect gene regulation during the zebrafish maternal to zygotic transition by combining single cell RNA sequencing with RNA metabolic labeling and nucleotide conversion within zebrafish embryos. We decompose single cell transcriptomes into their new zygotic and old maternal mRNA components  and elicit critical information on gene regulation as it unfolds over both time and space. We show that most cell type restricted expression arises by zygotic transcription  but distinguish a specific role for maternal transcripts in defining germ cell and enveloping layer identity  two earliest specified cell identities. We recover the underlying replacement between maternal and zygotic copies of embryonic genes with a relatively constant overall mRNA level  and associate a fast replacement with genes that has a restricted zygotic expression in either cell type or time. Our study provides a valuable resource to investigate maternal and zygotic transcriptomes and reveals post transcriptional events that control gene regulation during early embryogenesis. Overall design: single cell  SLAM seq  3 developmental stages  with treated and untreated samples  and biological replicates,parent bioproject:PRJNA933118,pubmed:37131717;pubmed:38600066,,zebrafish embryo  50% epiboly  NT  replicate B  scRNAseq,GSM7035744,,source name:zebrafish embryo|tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:NT|geo loc name:missing|collection date:missing,zebrafish embryo  50% epiboly  NT  replicate B  scRNAseq,Alignment of sequencing reads and generation of digital expression matrices was performed essentially as described in Farrell et. al.  Science  2018  using Drop seq tools v1.12. Two modified versions of the genome were produced: one where all T residues were replaced with C and one where all A residues were replaced with G. Prior to alignment  original sequencing reads were preserved and then all A residues were replaced with G. These converted reads were aligned to both modified genomes using Bowtie2 as previously described  then filtered based on which strand the read was aligned to. These two outputs were combined and then processed through the remainder of the Drop seq tools pipeline to produce processed BAM files and digital gene expression matrices. Converted reads were then replaced with the original reads and CIGAR strings were recalculated using Samtools in the final output BAM files. Assembly: GRCz11/102 Supplementary files format and content: Tab separated values files and matrix files,zebrafish embryo,20mM 4sUTP injection at 1 cell stage,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:NT,GSM7035744,GSM7035744: zebrafish embryo  50% epiboly  NT  replicate B  scRNAseq; Danio rerio; RNA Seq,GSM7035744 r1,GSM7035744,1,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP422528,,loader:fastq load.py,B09_S9_I1_001.fastq.gz B09_S9_R1_001.fastq.gz B09_S9_R2_001.fastq.gz,fastq fastq fastq,1731750720.0,10308040.0,GSM7035744 r1,0:8 1:20 2:140,A:483160966;C:257831212;G:374014730;T:328009839;N:108853,8,20,140,,483160966,257831212,374014730,328009839,108853,SRX19321399,SRS16719697,SRA1590430,Hebrew University of Jerusalem,Hebrew University of Jerusalem,1,0.76439,,0.04865,,0.85794,,0.69865,,140,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,dropseq,,Israel,2023-02-09,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
74106,SRR23380801,SRX19321398,SRS16719698,SRP422528,PRJNA934545,A single cell map of maternal and zygotic mRNA dynamics during cell type specification in zebrafish embryos [scSLAM seq],GSE224918,Other,During early embryogenesis  embryos undergo a massive degradation of maternally inherited mRNAs and produce new zygotic transcripts. This maternal to zygotic transition requires a tight interplay of mRNA transcription and degradation  but distinguishing their unique contributions remains a challenge. Here  we dissect gene regulation during the zebrafish maternal to zygotic transition by combining single cell RNA sequencing with RNA metabolic labeling and nucleotide conversion within zebrafish embryos. We decompose single cell transcriptomes into their new zygotic and old maternal mRNA components  and elicit critical information on gene regulation as it unfolds over both time and space. We show that most cell type restricted expression arises by zygotic transcription  but distinguish a specific role for maternal transcripts in defining germ cell and enveloping layer identity  two earliest specified cell identities. We recover the underlying replacement between maternal and zygotic copies of embryonic genes with a relatively constant overall mRNA level  and associate a fast replacement with genes that has a restricted zygotic expression in either cell type or time. Our study provides a valuable resource to investigate maternal and zygotic transcriptomes and reveals post transcriptional events that control gene regulation during early embryogenesis. Overall design: single cell  SLAM seq  3 developmental stages  with treated and untreated samples  and biological replicates,parent bioproject:PRJNA933118,pubmed:37131717;pubmed:38600066,,zebrafish embryo  50% epiboly  IAA  replicate B  scRNAseq,GSM7035743,,source name:zebrafish embryo|tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:IAA|geo loc name:missing|collection date:missing,zebrafish embryo  50% epiboly  IAA  replicate B  scRNAseq,Alignment of sequencing reads and generation of digital expression matrices was performed essentially as described in Farrell et. al.  Science  2018  using Drop seq tools v1.12. Two modified versions of the genome were produced: one where all T residues were replaced with C and one where all A residues were replaced with G. Prior to alignment  original sequencing reads were preserved and then all A residues were replaced with G. These converted reads were aligned to both modified genomes using Bowtie2 as previously described  then filtered based on which strand the read was aligned to. These two outputs were combined and then processed through the remainder of the Drop seq tools pipeline to produce processed BAM files and digital gene expression matrices. Converted reads were then replaced with the original reads and CIGAR strings were recalculated using Samtools in the final output BAM files. Assembly: GRCz11/102 Supplementary files format and content: Tab separated values files and matrix files,zebrafish embryo,20mM 4sUTP injection at 1 cell stage,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:IAA,GSM7035743,GSM7035743: zebrafish embryo  50% epiboly  IAA  replicate B  scRNAseq; Danio rerio; RNA Seq,GSM7035743 r1,GSM7035743,1,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP422528,,loader:fastq load.py,B10_S10_I1_001.fastq.gz B10_S10_R1_001.fastq.gz B10_S10_R2_001.fastq.gz,fastq fastq fastq,1449614208.0,8628656.0,GSM7035743 r1,0:8 1:20 2:140,A:383054921;C:231791450;G:292787007;T:300288679;N:89783,8,20,140,,383054921,231791450,292787007,300288679,89783,SRX19321398,SRS16719698,SRA1590430,Hebrew University of Jerusalem,Hebrew University of Jerusalem,1,0.69705,,0.03055,,0.86009,,0.49815,,140,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,dropseq,,Israel,2023-02-09,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
74107,SRR23380802,SRX19321397,SRS16719696,SRP422528,PRJNA934545,A single cell map of maternal and zygotic mRNA dynamics during cell type specification in zebrafish embryos [scSLAM seq],GSE224918,Other,During early embryogenesis  embryos undergo a massive degradation of maternally inherited mRNAs and produce new zygotic transcripts. This maternal to zygotic transition requires a tight interplay of mRNA transcription and degradation  but distinguishing their unique contributions remains a challenge. Here  we dissect gene regulation during the zebrafish maternal to zygotic transition by combining single cell RNA sequencing with RNA metabolic labeling and nucleotide conversion within zebrafish embryos. We decompose single cell transcriptomes into their new zygotic and old maternal mRNA components  and elicit critical information on gene regulation as it unfolds over both time and space. We show that most cell type restricted expression arises by zygotic transcription  but distinguish a specific role for maternal transcripts in defining germ cell and enveloping layer identity  two earliest specified cell identities. We recover the underlying replacement between maternal and zygotic copies of embryonic genes with a relatively constant overall mRNA level  and associate a fast replacement with genes that has a restricted zygotic expression in either cell type or time. Our study provides a valuable resource to investigate maternal and zygotic transcriptomes and reveals post transcriptional events that control gene regulation during early embryogenesis. Overall design: single cell  SLAM seq  3 developmental stages  with treated and untreated samples  and biological replicates,parent bioproject:PRJNA933118,pubmed:37131717;pubmed:38600066,,zebrafish embryo  50% epiboly  NT  replicate A  scRNAseq,GSM7035742,,source name:zebrafish embryo|tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:NT|geo loc name:missing|collection date:missing,zebrafish embryo  50% epiboly  NT  replicate A  scRNAseq,Alignment of sequencing reads and generation of digital expression matrices was performed essentially as described in Farrell et. al.  Science  2018  using Drop seq tools v1.12. Two modified versions of the genome were produced: one where all T residues were replaced with C and one where all A residues were replaced with G. Prior to alignment  original sequencing reads were preserved and then all A residues were replaced with G. These converted reads were aligned to both modified genomes using Bowtie2 as previously described  then filtered based on which strand the read was aligned to. These two outputs were combined and then processed through the remainder of the Drop seq tools pipeline to produce processed BAM files and digital gene expression matrices. Converted reads were then replaced with the original reads and CIGAR strings were recalculated using Samtools in the final output BAM files. Assembly: GRCz11/102 Supplementary files format and content: Tab separated values files and matrix files,zebrafish embryo,20mM 4sUTP injection at 1 cell stage,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:NT,GSM7035742,GSM7035742: zebrafish embryo  50% epiboly  NT  replicate A  scRNAseq; Danio rerio; RNA Seq,GSM7035742 r1,GSM7035742,1,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP422528,,loader:fastq load.py,B03_S3_I1_001.fastq.gz B03_S3_R1_001.fastq.gz B03_S3_R2_001.fastq.gz,fastq fastq fastq,3289212696.0,19578647.0,GSM7035742 r1,0:8 1:20 2:140,A:917014925;C:511489537;G:636621002;T:675689845;N:195271,8,20,140,,917014925,511489537,636621002,675689845,195271,SRX19321397,SRS16719696,SRA1590430,Hebrew University of Jerusalem,Hebrew University of Jerusalem,1,0.84983,,0.0403,,0.84948,,0.66672,,140,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,dropseq,,Israel,2023-02-09,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
74108,SRR23380803,SRX19321396,SRS16719695,SRP422528,PRJNA934545,A single cell map of maternal and zygotic mRNA dynamics during cell type specification in zebrafish embryos [scSLAM seq],GSE224918,Other,During early embryogenesis  embryos undergo a massive degradation of maternally inherited mRNAs and produce new zygotic transcripts. This maternal to zygotic transition requires a tight interplay of mRNA transcription and degradation  but distinguishing their unique contributions remains a challenge. Here  we dissect gene regulation during the zebrafish maternal to zygotic transition by combining single cell RNA sequencing with RNA metabolic labeling and nucleotide conversion within zebrafish embryos. We decompose single cell transcriptomes into their new zygotic and old maternal mRNA components  and elicit critical information on gene regulation as it unfolds over both time and space. We show that most cell type restricted expression arises by zygotic transcription  but distinguish a specific role for maternal transcripts in defining germ cell and enveloping layer identity  two earliest specified cell identities. We recover the underlying replacement between maternal and zygotic copies of embryonic genes with a relatively constant overall mRNA level  and associate a fast replacement with genes that has a restricted zygotic expression in either cell type or time. Our study provides a valuable resource to investigate maternal and zygotic transcriptomes and reveals post transcriptional events that control gene regulation during early embryogenesis. Overall design: single cell  SLAM seq  3 developmental stages  with treated and untreated samples  and biological replicates,parent bioproject:PRJNA933118,pubmed:37131717;pubmed:38600066,,zebrafish embryo  50% epiboly  IAA  replicate A  scRNAseq,GSM7035741,,source name:zebrafish embryo|tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:IAA|geo loc name:missing|collection date:missing,zebrafish embryo  50% epiboly  IAA  replicate A  scRNAseq,Alignment of sequencing reads and generation of digital expression matrices was performed essentially as described in Farrell et. al.  Science  2018  using Drop seq tools v1.12. Two modified versions of the genome were produced: one where all T residues were replaced with C and one where all A residues were replaced with G. Prior to alignment  original sequencing reads were preserved and then all A residues were replaced with G. These converted reads were aligned to both modified genomes using Bowtie2 as previously described  then filtered based on which strand the read was aligned to. These two outputs were combined and then processed through the remainder of the Drop seq tools pipeline to produce processed BAM files and digital gene expression matrices. Converted reads were then replaced with the original reads and CIGAR strings were recalculated using Samtools in the final output BAM files. Assembly: GRCz11/102 Supplementary files format and content: Tab separated values files and matrix files,zebrafish embryo,20mM 4sUTP injection at 1 cell stage,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,tissue:zebrafish embryo|developmental stage:50% epiboly|strain:AB/TL|treatment:IAA,GSM7035741,GSM7035741: zebrafish embryo  50% epiboly  IAA  replicate A  scRNAseq; Danio rerio; RNA Seq,GSM7035741 r1,GSM7035741,1,Drop seq droplet encapsulation of cells. Libraries were built according to the Drop seq protocol version 3.1 12/28/2015  available http://www.dropseq.org/ with the following modifications. Iodoacetamide IAA  Sigma Aldrich was diluted in ethanol to a 100mM working concentration. Following breakage of droplets  beads were washed once with 300uL of 5x IAA buffer 250mM NaPO4 pH8  and incubated in IAA solution 50mM NaPO4 pH8  10mM IAA  20% DMSO  6% Ficoll PM 400 for 15min at 32°C with rotation. DTT was added to a final concentration of 20mM to stop the reaction  and beads were washed twice with 6x SSC solution before continuing with reverse transcription. An estimated 50–100 STAMPs were included in each 50uL PCR reaction  and 12–13 cycles of PCR amplification were performed. Libraries were sequenced using Illumina Nextseq v2.2 Mid or High Output 150bp chemistry.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP422528,,loader:fastq load.py,B04_S4_R1_001.fastq.gz B04_S4_R2_001.fastq.gz B04_S4_I1_001.fastq.gz,fastq fastq fastq,18799793040.0,111903530.0,GSM7035741 r1,0:8 1:20 2:140,A:5320607436;C:2779767285;G:3920270375;T:3644700536;N:1148568,8,20,140,,5320607436,2779767285,3920270375,3644700536,1148568,SRX19321396,SRS16719695,SRA1590430,Hebrew University of Jerusalem,Hebrew University of Jerusalem,1,0.64644,,0.0354,,0.85886,,0.48293,,140,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,dropseq,,Israel,2023-02-09,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
74371,SRR23691702,SRX19554125,SRS16938871,SRP425398,PRJNA940501,Zebrahub: Multimodal Zebrafish Developmental Atlas,PRJNA940501,Other,A single embryo single cell RNA seq developmental atlas of Zebrafish.,,,,TDR24,TDR24 budstage EKW NA none 10x,,strain:EKW|isolate:single embryo|breed:siblings per timepoint|cultivar:not applicable|ecotype:not applicable|age:10 hpf|sex:not determined|tissue:Whole embryo|replicate:rep2|BioSampleModel:Model organism or animal,,,,,,,,,TDR24,TDR24 budstage EKW NA none 10x,TDR24 budstage EKW NA none 10x,single embryo dissociation,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP425398,,,TDR24_budstage_EKW_NA_none_10x_S2_L001_I1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L001_R1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L001_R2_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L002_I1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L002_R1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L002_R2_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L003_I1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L003_R1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L003_R2_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L004_I1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L004_R1_001.fastq.gz TDR24_budstage_EKW_NA_none_10x_S2_L004_R2_001.fastq.gz,fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq,,,TDR24 budstage EKW NA none 10x S2 L001 I1 001.fastq.gz,,,,,,,,,,,,SRX19554125,,SRA1598656,Chan Zuckerberg Biohub|Quantitative Cell Science,Chan Zuckerberg Biohub,,,,,,,,,,,,,,,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2023-03-02,Gastrula,Embryo,Whole Organism,All anatomical structures
74386,SRR23691699,SRX19554110,SRS16938856,SRP425398,PRJNA940501,Zebrahub: Multimodal Zebrafish Developmental Atlas,PRJNA940501,Other,A single embryo single cell RNA seq developmental atlas of Zebrafish.,,,,TDR25,TDR25 budstage EKW NA none 10x,,strain:EKW|isolate:single embryo|breed:siblings per timepoint|cultivar:not applicable|ecotype:not applicable|age:10 hpf|sex:not determined|tissue:Whole embryo|replicate:rep3|BioSampleModel:Model organism or animal,,,,,,,,,TDR25,TDR25 budstage EKW NA none 10x,TDR25 budstage EKW NA none 10x,single embryo dissociation,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP425398,,,TDR25_budstage_EKW_NA_none_10x_S3_L001_I1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L001_R1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L001_R2_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L002_I1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L002_R1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L002_R2_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L003_I1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L003_R1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L003_R2_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L004_I1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L004_R1_001.fastq.gz TDR25_budstage_EKW_NA_none_10x_S3_L004_R2_001.fastq.gz,fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq,,,TDR25 budstage EKW NA none 10x S3 L001 I1 001.fastq.gz,,,,,,,,,,,,SRX19554110,,SRA1598656,Chan Zuckerberg Biohub|Quantitative Cell Science,Chan Zuckerberg Biohub,,,,,,,,,,,,,,,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2023-03-02,Gastrula,Embryo,Whole Organism,All anatomical structures
74387,SRR23691701,SRX19554109,SRS16938855,SRP425398,PRJNA940501,Zebrahub: Multimodal Zebrafish Developmental Atlas,PRJNA940501,Other,A single embryo single cell RNA seq developmental atlas of Zebrafish.,,,,TDR23,TDR23 budstage EKW NA none 10x,,strain:EKW|isolate:single embryo|breed:siblings per timepoint|cultivar:not applicable|ecotype:not applicable|age:10 hpf|sex:not determined|tissue:Whole embryo|replicate:rep1|BioSampleModel:Model organism or animal,,,,,,,,,TDR23,TDR23 budstage EKW NA none 10x,TDR23 budstage EKW NA none 10x,single embryo dissociation,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP425398,,,TDR23_budstage_EKW_NA_none_10x_S1_L001_I1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L001_R1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L001_R2_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L002_I1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L002_R1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L002_R2_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L003_I1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L003_R1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L003_R2_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L004_I1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L004_R1_001.fastq.gz TDR23_budstage_EKW_NA_none_10x_S1_L004_R2_001.fastq.gz,fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq,,,TDR23 budstage EKW NA none 10x S1 L001 I1 001.fastq.gz,,,,,,,,,,,,SRX19554109,,SRA1598656,Chan Zuckerberg Biohub|Quantitative Cell Science,Chan Zuckerberg Biohub,,,,,,,,,,,,,,,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2023-03-02,Gastrula,Embryo,Whole Organism,All anatomical structures
74402,SRR23691700,SRX19554094,SRS16938840,SRP425398,PRJNA940501,Zebrahub: Multimodal Zebrafish Developmental Atlas,PRJNA940501,Other,A single embryo single cell RNA seq developmental atlas of Zebrafish.,,,,TDR26,TDR26 budstage EKW NA none 10x,,strain:EKW|isolate:single embryo|breed:siblings per timepoint|cultivar:not applicable|ecotype:not applicable|age:10 hpf|sex:not determined|tissue:Whole embryo|replicate:rep4|BioSampleModel:Model organism or animal,,,,,,,,,TDR26,TDR26 budstage EKW NA none 10x,TDR26 budstage EKW NA none 10x,single embryo dissociation,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,PAIRED,ILLUMINA,Illumina NovaSeq 6000,,SRP425398,,,TDR26_budstage_EKW_NA_none_10x_S4_L001_I1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L001_R1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L001_R2_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L002_I1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L002_R1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L002_R2_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L003_I1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L003_R1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L003_R2_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L004_I1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L004_R1_001.fastq.gz TDR26_budstage_EKW_NA_none_10x_S4_L004_R2_001.fastq.gz,fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq fastq,,,TDR26 budstage EKW NA none 10x S4 L001 I1 001.fastq.gz,,,,,,,,,,,,SRX19554094,,SRA1598656,Chan Zuckerberg Biohub|Quantitative Cell Science,Chan Zuckerberg Biohub,,,,,,,,,,,,,,,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2023-03-02,Gastrula,Embryo,Whole Organism,All anatomical structures