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
7953,ERR2146982,ERX2203515,ERS1952873,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY control6,SAMEA104327895,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327895|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:23Z|INSDC status:public|Submitter Id:E MTAB 6077:XY control6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP negative|sample name:E MTAB 6077:XY control6|well information:4000 cells tube control,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY control6 p,XY control6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP negative|Experimental Factor: single cell identifier:XY control6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_control6_R2.fastq.gz XY_control6_R1.fastq.gz,fastq fastq,4549556088.0,18053794.0,E MTAB 6077:XY control6 R,0:126 1:126,A:1249771585;C:1032539820;G:1013780979;T:1253138208;N:325496,126,126,,,1249771585,1032539820,1013780979,1253138208,325496,ERX2203515,ERS1952873,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.93704,0.93646,0.09456,0.0963,0.77989,0.78277,0.49027,0.49534,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7954,ERR2146981,ERX2203514,ERS1952872,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY control5,SAMEA104327894,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327894|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY control5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|sample name:E MTAB 6077:XY control5|well information:4000 cells tube control,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY control5 p,XY control5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY control5|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_control5_R1.fastq.gz XY_control5_R2.fastq.gz,fastq fastq,4716306756.0,18715503.0,E MTAB 6077:XY control5 R,0:126 1:126,A:1270474643;C:1095125173;G:1073972754;T:1276395367;N:338819,126,126,,,1270474643,1095125173,1073972754,1276395367,338819,ERX2203514,ERS1952872,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.93411,0.93498,0.08638,0.08804,0.77893,0.78094,0.48544,0.49009,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7955,ERR2146980,ERX2203513,ERS1952871,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY control4,SAMEA104327893,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327893|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY control4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP negative|sample name:E MTAB 6077:XY control4|well information:4000 cells tube control,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY control4 p,XY control4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP negative|Experimental Factor: single cell identifier:XY control4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_control4_R1.fastq.gz XY_control4_R2.fastq.gz,fastq fastq,5945132844.0,23591797.0,E MTAB 6077:XY control4 R,0:126 1:126,A:1626789290;C:1357026290;G:1322234145;T:1638578806;N:504313,126,126,,,1626789290,1357026290,1322234145,1638578806,504313,ERX2203513,ERS1952871,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.92339,0.92233,0.10762,0.11113,0.77218,0.77833,0.48434,0.47741,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7956,ERR2146979,ERX2203512,ERS1952870,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY control3,SAMEA104327892,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327892|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY control3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|sample name:E MTAB 6077:XY control3|well information:4000 cells tube control,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY control3 p,XY control3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY control3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_control3_R1.fastq.gz XY_control3_R2.fastq.gz,fastq fastq,5535067608.0,21964554.0,E MTAB 6077:XY control3 R,0:126 1:126,A:1514179525;C:1264413791;G:1243494617;T:1512516950;N:462725,126,126,,,1514179525,1264413791,1243494617,1512516950,462725,ERX2203512,ERS1952870,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.91933,0.9182,0.10843,0.11009,0.77662,0.77839,0.48196,0.48344,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7957,ERR2146978,ERX2203511,ERS1952869,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY control2,SAMEA104327891,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327891|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY control2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP negative|sample name:E MTAB 6077:XY control2|well information:4000 cells tube control,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY control2 p,XY control2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP negative|Experimental Factor: single cell identifier:XY control2|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_control2_R1.fastq.gz XY_control2_R2.fastq.gz,fastq fastq,5765575536.0,22879268.0,E MTAB 6077:XY control2 R,0:126 1:126,A:1577072713;C:1319503344;G:1284653366;T:1583881864;N:464249,126,126,,,1577072713,1319503344,1284653366,1583881864,464249,ERX2203511,ERS1952869,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.90176,0.90134,0.09386,0.09519,0.78654,0.7875,0.48786,0.48901,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7958,ERR2146977,ERX2203510,ERS1952868,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY control1,SAMEA104327890,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327890|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY control1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|sample name:E MTAB 6077:XY control1|well information:4000 cells tube control,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY control1 p,XY control1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY control1|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_control1_R1.fastq.gz XY_control1_R2.fastq.gz,fastq fastq,5364104256.0,21286128.0,E MTAB 6077:XY control1 R,0:126 1:126,A:1451397579;C:1239771441;G:1215928457;T:1456576239;N:430540,126,126,,,1451397579,1239771441,1215928457,1456576239,430540,ERX2203510,ERS1952868,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87836,0.87758,0.09375,0.09537,0.79101,0.79385,0.48123,0.48531,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7959,ERR2146976,ERX2203509,ERS1952867,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H8,SAMEA104327889,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327889|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H8 p,XY H8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H8_R1.fastq.gz XY_H8_R2.fastq.gz,fastq fastq,864210816.0,3429408.0,E MTAB 6077:XY H8 R,0:126 1:126,A:249771934;C:187002108;G:178230223;T:249134097;N:72454,126,126,,,249771934,187002108,178230223,249134097,72454,ERX2203509,ERS1952867,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81405,0.81553,0.21726,0.2187,0.94909,0.94913,0.762,0.75712,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7960,ERR2146975,ERX2203508,ERS1952866,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H7,SAMEA104327888,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327888|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H7 p,XY H7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H7_R1.fastq.gz XY_H7_R2.fastq.gz,fastq fastq,756021672.0,3000086.0,E MTAB 6077:XY H7 R,0:126 1:126,A:218450821;C:163414581;G:153421674;T:220674025;N:60571,126,126,,,218450821,163414581,153421674,220674025,60571,ERX2203508,ERS1952866,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.86606,0.86563,0.20652,0.20672,0.90678,0.90684,0.52647,0.52546,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7961,ERR2146974,ERX2203507,ERS1952865,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H6,SAMEA104327887,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327887|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H6 p,XY H6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H6_R1.fastq.gz XY_H6_R2.fastq.gz,fastq fastq,1074550176.0,4264088.0,E MTAB 6077:XY H6 R,0:126 1:126,A:308254957;C:234011984;G:222627836;T:309568320;N:87079,126,126,,,308254957,234011984,222627836,309568320,87079,ERX2203507,ERS1952865,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.83064,0.8297,0.17609,0.17705,0.94706,0.94688,0.70976,0.71858,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7962,ERR2146973,ERX2203506,ERS1952864,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G7,SAMEA104327886,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327886|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G7 p,XY G7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G7_R1.fastq.gz XY_G7_R2.fastq.gz,fastq fastq,888876576.0,3527288.0,E MTAB 6077:XY G7 R,0:126 1:126,A:254439819;C:194980753;G:181687161;T:257695391;N:73452,126,126,,,254439819,194980753,181687161,257695391,73452,ERX2203506,ERS1952864,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.90072,0.89984,0.2275,0.22842,0.91116,0.91165,0.56832,0.5707,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7963,ERR2146972,ERX2203505,ERS1952863,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G6,SAMEA104327885,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327885|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G6 p,XY G6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G6_R1.fastq.gz XY_G6_R2.fastq.gz,fastq fastq,996116436.0,3952843.0,E MTAB 6077:XY G6 R,0:126 1:126,A:275654881;C:226382568;G:215845958;T:278153726;N:79303,126,126,,,275654881,226382568,215845958,278153726,79303,ERX2203505,ERS1952863,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.91202,0.91213,0.07492,0.07611,0.93454,0.93446,0.48374,0.48865,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7964,ERR2146971,ERX2203504,ERS1952862,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F8,SAMEA104327884,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327884|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F8 p,XY F8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F8_R1.fastq.gz XY_F8_R2.fastq.gz,fastq fastq,877436028.0,3481889.0,E MTAB 6077:XY F8 R,0:126 1:126,A:241235862;C:201062056;G:193297643;T:241769388;N:71079,126,126,,,241235862,201062056,193297643,241769388,71079,ERX2203504,ERS1952862,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80726,0.80669,0.05191,0.05278,0.9447,0.94481,0.4442,0.44721,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7965,ERR2146970,ERX2203503,ERS1952861,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F7,SAMEA104327883,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327883|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F7 p,XY F7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F7_R1.fastq.gz XY_F7_R2.fastq.gz,fastq fastq,958632192.0,3804096.0,E MTAB 6077:XY F7 R,0:126 1:126,A:273424334;C:210648698;G:198846984;T:275634740;N:77436,126,126,,,273424334,210648698,198846984,275634740,77436,ERX2203503,ERS1952861,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.90705,0.90578,0.17689,0.17782,0.90224,0.90258,0.52906,0.52965,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7966,ERR2146969,ERX2203502,ERS1952860,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F6,SAMEA104327882,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327882|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F6 p,XY F6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F6_R1.fastq.gz XY_F6_R2.fastq.gz,fastq fastq,819905940.0,3253595.0,E MTAB 6077:XY F6 R,0:126 1:126,A:235169787;C:178657976;G:168253811;T:237755989;N:68377,126,126,,,235169787,178657976,168253811,237755989,68377,ERX2203502,ERS1952860,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.8318,0.83391,0.25866,0.26072,0.93363,0.93413,0.64306,0.64461,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7967,ERR2146968,ERX2203501,ERS1952859,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E8,SAMEA104327881,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327881|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E8 p,XY E8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E8_R1.fastq.gz XY_E8_R2.fastq.gz,fastq fastq,842724540.0,3344145.0,E MTAB 6077:XY E8 R,0:126 1:126,A:242981289;C:183320053;G:172749908;T:243603429;N:69861,126,126,,,242981289,183320053,172749908,243603429,69861,ERX2203501,ERS1952859,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81153,0.81108,0.25264,0.25373,0.95215,0.95243,0.7481,0.748,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7968,ERR2146967,ERX2203500,ERS1952858,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E7,SAMEA104327880,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327880|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E7 p,XY E7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E7_R1.fastq.gz XY_E7_R2.fastq.gz,fastq fastq,776402424.0,3080962.0,E MTAB 6077:XY E7 R,0:126 1:126,A:219057494;C:173933939;G:161687576;T:221658032;N:65383,126,126,,,219057494,173933939,161687576,221658032,65383,ERX2203500,ERS1952858,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81289,0.8127,0.17319,0.17455,0.93941,0.94002,0.61509,0.61689,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7969,ERR2146966,ERX2203499,ERS1952857,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E6,SAMEA104327879,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327879|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E6 p,XY E6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E6_R1.fastq.gz XY_E6_R2.fastq.gz,fastq fastq,857047464.0,3400982.0,E MTAB 6077:XY E6 R,0:126 1:126,A:241359294;C:190499276;G:181314236;T:243803691;N:70967,126,126,,,241359294,190499276,181314236,243803691,70967,ERX2203499,ERS1952857,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87204,0.87145,0.14714,0.1482,0.92618,0.92654,0.51966,0.40678,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7970,ERR2146965,ERX2203498,ERS1952856,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D8,SAMEA104327878,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327878|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D8 p,XY D8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D8_R1.fastq.gz XY_D8_R2.fastq.gz,fastq fastq,843642324.0,3347787.0,E MTAB 6077:XY D8 R,0:126 1:126,A:242903477;C:184375610;G:173102506;T:243189515;N:71216,126,126,,,242903477,184375610,173102506,243189515,71216,ERX2203498,ERS1952856,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80439,0.80704,0.25569,0.25887,0.95485,0.9554,0.77117,0.77377,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7971,ERR2146964,ERX2203497,ERS1952855,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D7,SAMEA104327877,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327877|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D7 p,XY D7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D7_R1.fastq.gz XY_D7_R2.fastq.gz,fastq fastq,984207168.0,3905584.0,E MTAB 6077:XY D7 R,0:126 1:126,A:283251031;C:217529580;G:197592673;T:285746776;N:87108,126,126,,,283251031,217529580,197592673,285746776,87108,ERX2203497,ERS1952855,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85387,0.85464,0.19987,0.20244,0.94343,0.94416,0.76877,0.76643,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7972,ERR2146963,ERX2203496,ERS1952854,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D6,SAMEA104327876,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327876|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D6 p,XY D6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D6_R1.fastq.gz XY_D6_R2.fastq.gz,fastq fastq,822279780.0,3263015.0,E MTAB 6077:XY D6 R,0:126 1:126,A:235190518;C:179206449;G:169869879;T:237947879;N:65055,126,126,,,235190518,179206449,169869879,237947879,65055,ERX2203496,ERS1952854,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87845,0.88077,0.13151,0.13349,0.91488,0.915,0.58175,0.58204,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7973,ERR2146962,ERX2203495,ERS1952853,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C8,SAMEA104327875,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327875|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C8 p,XY C8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C8_R1.fastq.gz XY_C8_R2.fastq.gz,fastq fastq,1013469408.0,4021704.0,E MTAB 6077:XY C8 R,0:126 1:126,A:292880928;C:221164286;G:209883396;T:289460688;N:80110,126,126,,,292880928,221164286,209883396,289460688,80110,ERX2203495,ERS1952853,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.83814,0.8391,0.24905,0.24961,0.93736,0.93667,0.61348,0.61766,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7974,ERR2146961,ERX2203494,ERS1952852,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C7,SAMEA104327874,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327874|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C7 p,XY C7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C7_R1.fastq.gz XY_C7_R2.fastq.gz,fastq fastq,1006828704.0,3995352.0,E MTAB 6077:XY C7 R,0:126 1:126,A:287564865;C:223387104;G:208613518;T:287183841;N:79376,126,126,,,287564865,223387104,208613518,287183841,79376,ERX2203494,ERS1952852,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87244,0.87249,0.16434,0.16605,0.91597,0.91687,0.55002,0.54756,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7975,ERR2146960,ERX2203493,ERS1952851,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C6,SAMEA104327873,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327873|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C6 p,XY C6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C6_R1.fastq.gz XY_C6_R2.fastq.gz,fastq fastq,961789500.0,3816625.0,E MTAB 6077:XY C6 R,0:126 1:126,A:278781495;C:205421491;G:197677532;T:279831229;N:77753,126,126,,,278781495,205421491,197677532,279831229,77753,ERX2203493,ERS1952851,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.90742,0.90809,0.18725,0.18852,0.9068,0.90642,0.54244,0.46695,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7976,ERR2146959,ERX2203492,ERS1952850,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B8,SAMEA104327872,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327872|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B8 p,XY B8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B8_R1.fastq.gz XY_B8_R2.fastq.gz,fastq fastq,919461816.0,3648658.0,E MTAB 6077:XY B8 R,0:126 1:126,A:269811899;C:193898193;G:185943309;T:269733141;N:75274,126,126,,,269811899,193898193,185943309,269733141,75274,ERX2203492,ERS1952850,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85046,0.8503,0.27069,0.27204,0.91944,0.92046,0.54761,0.54669,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7977,ERR2146958,ERX2203491,ERS1952849,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B7,SAMEA104327871,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327871|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B7 p,XY B7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B7_R1.fastq.gz XY_B7_R2.fastq.gz,fastq fastq,1105211268.0,4385759.0,E MTAB 6077:XY B7 R,0:126 1:126,A:319612550;C:237937703;G:225942677;T:321629855;N:88483,126,126,,,319612550,237937703,225942677,321629855,88483,ERX2203491,ERS1952849,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.89722,0.89732,0.2253,0.22622,0.9223,0.92214,0.46651,0.47996,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7978,ERR2146957,ERX2203490,ERS1952848,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B6,SAMEA104327870,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327870|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B6 p,XY B6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B6|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B6_R1.fastq.gz XY_B6_R2.fastq.gz,fastq fastq,888803496.0,3526998.0,E MTAB 6077:XY B6 R,0:126 1:126,A:259157968;C:187867811;G:181734930;T:259972893;N:69894,126,126,,,259157968,187867811,181734930,259972893,69894,ERX2203490,ERS1952848,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.905,0.90517,0.14694,0.14784,0.90173,0.90205,0.5606,0.53635,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7979,ERR2146956,ERX2203489,ERS1952847,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A9,SAMEA104327869,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327869|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A9 p,XY A9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A9|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A9_R1.fastq.gz XY_A9_R2.fastq.gz,fastq fastq,852383952.0,3382476.0,E MTAB 6077:XY A9 R,0:126 1:126,A:243063922;C:186833267;G:175428225;T:246986620;N:71918,126,126,,,243063922,186833267,175428225,246986620,71918,ERX2203489,ERS1952847,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.791,0.79236,0.26001,0.26219,0.95495,0.95515,0.67722,0.67292,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7980,ERR2146955,ERX2203488,ERS1952846,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A8,SAMEA104327868,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327868|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A8 p,XY A8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A8_R1.fastq.gz XY_A8_R2.fastq.gz,fastq fastq,930505212.0,3692481.0,E MTAB 6077:XY A8 R,0:126 1:126,A:265557007;C:204874994;G:193354741;T:266645204;N:73266,126,126,,,265557007,204874994,193354741,266645204,73266,ERX2203488,ERS1952846,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80883,0.81059,0.25464,0.25659,0.95024,0.95051,0.75494,0.76047,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7981,ERR2146954,ERX2203487,ERS1952845,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A7,SAMEA104327867,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327867|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A7|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A7|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A7 p,XY A7 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A7|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A7_R1.fastq.gz XY_A7_R2.fastq.gz,fastq fastq,947373588.0,3759419.0,E MTAB 6077:XY A7 R,0:126 1:126,A:269034866;C:210414489;G:195690143;T:272157870;N:76220,126,126,,,269034866,210414489,195690143,272157870,76220,ERX2203487,ERS1952845,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.90451,0.90515,0.17025,0.17226,0.90774,0.90776,0.59799,0.60776,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7982,ERR2146953,ERX2203486,ERS1952844,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H5,SAMEA104327866,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327866|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H5 p,XY H5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H5_R1.fastq.gz XY_H5_R2.fastq.gz,fastq fastq,661968468.0,2626859.0,E MTAB 6077:XY H5 R,0:126 1:126,A:182197480;C:151742936;G:143603852;T:184373864;N:50336,126,126,,,182197480,151742936,143603852,184373864,50336,ERX2203486,ERS1952844,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.43701,0.45028,0.1003,0.10327,0.94828,0.94809,0.66076,0.66111,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7983,ERR2146952,ERX2203485,ERS1952843,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H4,SAMEA104327865,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327865|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H4 p,XY H4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H4_R1.fastq.gz XY_H4_R2.fastq.gz,fastq fastq,763960932.0,3031591.0,E MTAB 6077:XY H4 R,0:126 1:126,A:210454823;C:173613405;G:168163464;T:211676418;N:52822,126,126,,,210454823,173613405,168163464,211676418,52822,ERX2203485,ERS1952843,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.88746,0.88783,0.1341,0.13524,0.89613,0.8966,0.41929,0.50102,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7984,ERR2146951,ERX2203484,ERS1952842,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H3,SAMEA104327864,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327864|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H3 p,XY H3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H3_R1.fastq.gz XY_H3_R2.fastq.gz,fastq fastq,659294244.0,2616247.0,E MTAB 6077:XY H3 R,0:126 1:126,A:188850616;C:143711118;G:137850770;T:188835454;N:46286,126,126,,,188850616,143711118,137850770,188835454,46286,ERX2203484,ERS1952842,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80539,0.80566,0.23678,0.23789,0.92013,0.92086,0.63014,0.63224,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7985,ERR2146950,ERX2203483,ERS1952841,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H2,SAMEA104327863,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327863|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H2 p,XY H2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H2_R1.fastq.gz XY_H2_R2.fastq.gz,fastq fastq,800579556.0,3176903.0,E MTAB 6077:XY H2 R,0:126 1:126,A:223283547;C:180576112;G:171695116;T:224965999;N:58782,126,126,,,223283547,180576112,171695116,224965999,58782,ERX2203483,ERS1952841,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.49111,0.50915,0.12273,0.12834,0.93322,0.93324,0.63392,0.63061,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7986,ERR2146949,ERX2203482,ERS1952840,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H12,SAMEA104327862,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327862|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:22Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H12|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H12|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H12 p,XY H12 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H12|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H12_R1.fastq.gz XY_H12_R2.fastq.gz,fastq fastq,1012529952.0,4017976.0,E MTAB 6077:XY H12 R,0:126 1:126,A:296329041;C:212818812;G:204681970;T:298612968;N:87161,126,126,,,296329041,212818812,204681970,298612968,87161,ERX2203482,ERS1952840,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85208,0.85109,0.2762,0.2772,0.90857,0.9096,0.5945,0.59648,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7987,ERR2146948,ERX2203481,ERS1952839,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H1,SAMEA104327861,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327861|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H1 p,XY H1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H1_R1.fastq.gz XY_H1_R2.fastq.gz,fastq fastq,1013476716.0,4021733.0,E MTAB 6077:XY H1 R,0:126 1:126,A:285093595;C:224698069;G:216782708;T:286832156;N:70188,126,126,,,285093595,224698069,216782708,286832156,70188,ERX2203481,ERS1952839,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85277,0.85221,0.20343,0.20573,0.91727,0.91735,0.5053,0.51218,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7988,ERR2146947,ERX2203480,ERS1952838,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G5,SAMEA104327860,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327860|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G5 p,XY G5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G5_R1.fastq.gz XY_G5_R2.fastq.gz,fastq fastq,573735204.0,2276727.0,E MTAB 6077:XY G5 R,0:126 1:126,A:160393081;C:130403439;G:120151582;T:162737783;N:49319,126,126,,,160393081,130403439,120151582,162737783,49319,ERX2203480,ERS1952838,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.67173,0.67076,0.21171,0.21167,0.94113,0.94111,0.70844,0.70511,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7989,ERR2146946,ERX2203479,ERS1952837,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G4,SAMEA104327859,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327859|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G4 p,XY G4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G4_R1.fastq.gz XY_G4_R2.fastq.gz,fastq fastq,545927004.0,2166377.0,E MTAB 6077:XY G4 R,0:126 1:126,A:155307335;C:120841181;G:111873434;T:157868534;N:36520,126,126,,,155307335,120841181,111873434,157868534,36520,ERX2203479,ERS1952837,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.74895,0.75073,0.23536,0.23798,0.93166,0.93129,0.62288,0.63112,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7990,ERR2146945,ERX2203478,ERS1952836,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G3,SAMEA104327858,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327858|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G3 p,XY G3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G3_R1.fastq.gz XY_G3_R2.fastq.gz,fastq fastq,865575396.0,3434823.0,E MTAB 6077:XY G3 R,0:126 1:126,A:247249274;C:189448794;G:180244568;T:248572085;N:60675,126,126,,,247249274,189448794,180244568,248572085,60675,ERX2203478,ERS1952836,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85277,0.85221,0.24206,0.24438,0.9025,0.90327,0.54937,0.54992,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7991,ERR2146944,ERX2203477,ERS1952835,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G2,SAMEA104327857,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327857|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G2 p,XY G2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G2_R1.fastq.gz XY_G2_R2.fastq.gz,fastq fastq,896890428.0,3559089.0,E MTAB 6077:XY G2 R,0:126 1:126,A:253308278;C:199243055;G:188714589;T:255563791;N:60715,126,126,,,253308278,199243055,188714589,255563791,60715,ERX2203477,ERS1952835,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87194,0.87088,0.16803,0.17014,0.90358,0.90402,0.51979,0.52116,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7992,ERR2146943,ERX2203476,ERS1952834,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G12,SAMEA104327856,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327856|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G12|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G12|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G12 p,XY G12 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G12|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G12_R1.fastq.gz XY_G12_R2.fastq.gz,fastq fastq,1125374040.0,4465770.0,E MTAB 6077:XY G12 R,0:126 1:126,A:327288208;C:244109991;G:225102810;T:328783539;N:89492,126,126,,,327288208,244109991,225102810,328783539,89492,ERX2203476,ERS1952834,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81746,0.81706,0.23392,0.23534,0.90504,0.90542,0.57236,0.5639,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7993,ERR2146942,ERX2203475,ERS1952833,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G1,SAMEA104327855,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327855|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G1 p,XY G1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G1_R1.fastq.gz XY_G1_R2.fastq.gz,fastq fastq,1044608796.0,4145273.0,E MTAB 6077:XY G1 R,0:126 1:126,A:286857143;C:240643381;G:229200315;T:287832267;N:75690,126,126,,,286857143,240643381,229200315,287832267,75690,ERX2203475,ERS1952833,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80886,0.80723,0.05734,0.05871,0.95225,0.95195,0.45793,0.45613,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7994,ERR2146941,ERX2203474,ERS1952832,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F5,SAMEA104327854,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327854|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F5 p,XY F5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F5_R1.fastq.gz XY_F5_R2.fastq.gz,fastq fastq,711136440.0,2821970.0,E MTAB 6077:XY F5 R,0:126 1:126,A:204645320;C:153976075;G:145232961;T:207224102;N:57982,126,126,,,204645320,153976075,145232961,207224102,57982,ERX2203474,ERS1952832,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81603,0.81552,0.21691,0.21789,0.90546,0.90638,0.5987,0.59269,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7995,ERR2146940,ERX2203473,ERS1952831,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F4,SAMEA104327853,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327853|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F4 p,XY F4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F4_R1.fastq.gz XY_F4_R2.fastq.gz,fastq fastq,536057172.0,2127211.0,E MTAB 6077:XY F4 R,0:126 1:126,A:150969583;C:119522056;G:112226958;T:153300653;N:37922,126,126,,,150969583,119522056,112226958,153300653,37922,ERX2203473,ERS1952831,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.73615,0.73496,0.20708,0.20786,0.92772,0.92847,0.63249,0.63336,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7996,ERR2146939,ERX2203472,ERS1952830,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F3,SAMEA104327852,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327852|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F3 p,XY F3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F3_R1.fastq.gz XY_F3_R2.fastq.gz,fastq fastq,1011342024.0,4013262.0,E MTAB 6077:XY F3 R,0:126 1:126,A:290499568;C:219169313;G:209796959;T:291802472;N:73712,126,126,,,290499568,219169313,209796959,291802472,73712,ERX2203472,ERS1952830,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87586,0.87628,0.25771,0.25973,0.89895,0.89927,0.50683,0.49896,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7997,ERR2146938,ERX2203471,ERS1952829,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F2,SAMEA104327851,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327851|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F2 p,XY F2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F2_R1.fastq.gz XY_F2_R2.fastq.gz,fastq fastq,655849908.0,2602579.0,E MTAB 6077:XY F2 R,0:126 1:126,A:185384632;C:145678225;G:137663286;T:187075170;N:48595,126,126,,,185384632,145678225,137663286,187075170,48595,ERX2203471,ERS1952829,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.74833,0.74693,0.18103,0.18218,0.91977,0.92013,0.59183,0.58697,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7998,ERR2146937,ERX2203470,ERS1952828,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F12,SAMEA104327850,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327850|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F12|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F12|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F12 p,XY F12 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F12|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F12_R1.fastq.gz XY_F12_R2.fastq.gz,fastq fastq,600785892.0,2384071.0,E MTAB 6077:XY F12 R,0:126 1:126,A:168276037;C:142094582;G:130010572;T:160355651;N:49050,126,126,,,168276037,142094582,130010572,160355651,49050,ERX2203470,ERS1952828,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.73023,0.72607,0.13773,0.13694,0.92251,0.92194,0.61985,0.64885,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
7999,ERR2146936,ERX2203469,ERS1952827,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F1,SAMEA104327849,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327849|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F1 p,XY F1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F1_R1.fastq.gz XY_F1_R2.fastq.gz,fastq fastq,997460100.0,3958175.0,E MTAB 6077:XY F1 R,0:126 1:126,A:281570675;C:220590738;G:211855088;T:283372566;N:71033,126,126,,,281570675,220590738,211855088,283372566,71033,ERX2203469,ERS1952827,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.88181,0.88086,0.18012,0.182,0.90408,0.90392,0.46631,0.47014,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8000,ERR2146935,ERX2203468,ERS1952826,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E5,SAMEA104327848,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327848|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E5 p,XY E5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E5_R1.fastq.gz XY_E5_R2.fastq.gz,fastq fastq,432076680.0,1714590.0,E MTAB 6077:XY E5 R,0:126 1:126,A:121965441;C:95536720;G:89987057;T:124553150;N:34312,126,126,,,121965441,95536720,89987057,124553150,34312,ERX2203468,ERS1952826,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.76796,0.76857,0.18379,0.18457,0.92285,0.92273,0.67279,0.67103,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8001,ERR2146934,ERX2203467,ERS1952825,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E4,SAMEA104327847,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327847|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E4 p,XY E4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E4_R1.fastq.gz XY_E4_R2.fastq.gz,fastq fastq,936553212.0,3716481.0,E MTAB 6077:XY E4 R,0:126 1:126,A:263117120;C:208551987;G:198259840;T:266556826;N:67439,126,126,,,263117120,208551987,198259840,266556826,67439,ERX2203467,ERS1952825,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.86272,0.86395,0.18092,0.18393,0.91963,0.92009,0.48049,0.47567,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8002,ERR2146933,ERX2203466,ERS1952824,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E3,SAMEA104327846,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327846|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E3 p,XY E3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E3_R1.fastq.gz XY_E3_R2.fastq.gz,fastq fastq,722298276.0,2866263.0,E MTAB 6077:XY E3 R,0:126 1:126,A:205661883;C:158846036;G:151046574;T:206691454;N:52329,126,126,,,205661883,158846036,151046574,206691454,52329,ERX2203466,ERS1952824,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.8281,0.82888,0.227,0.22808,0.91151,0.91224,0.57671,0.56941,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8003,ERR2146932,ERX2203465,ERS1952823,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E2,SAMEA104327845,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327845|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E2 p,XY E2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E2_R1.fastq.gz XY_E2_R2.fastq.gz,fastq fastq,898576812.0,3565781.0,E MTAB 6077:XY E2 R,0:126 1:126,A:253404969;C:199204646;G:190410032;T:255492244;N:64921,126,126,,,253404969,199204646,190410032,255492244,64921,ERX2203465,ERS1952823,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.88647,0.88521,0.17368,0.17572,0.90567,0.9066,0.43691,0.51405,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8004,ERR2146931,ERX2203464,ERS1952822,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E12,SAMEA104327844,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327844|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E12|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E12|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E12 p,XY E12 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E12|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E12_R1.fastq.gz XY_E12_R2.fastq.gz,fastq fastq,1097535852.0,4355301.0,E MTAB 6077:XY E12 R,0:126 1:126,A:300243354;C:263382681;G:238183855;T:295635494;N:90468,126,126,,,300243354,263382681,238183855,295635494,90468,ERX2203464,ERS1952822,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.61006,0.606,0.14083,0.13917,0.92001,0.92078,0.6148,0.61556,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8005,ERR2146930,ERX2203463,ERS1952821,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E1,SAMEA104327843,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327843|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E1 p,XY E1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E1_R1.fastq.gz XY_E1_R2.fastq.gz,fastq fastq,746685072.0,2963036.0,E MTAB 6077:XY E1 R,0:126 1:126,A:211710079;C:165593782;G:155759006;T:213566590;N:55615,126,126,,,211710079,165593782,155759006,213566590,55615,ERX2203463,ERS1952821,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.75556,0.75733,0.19795,0.20011,0.91632,0.91672,0.60899,0.60441,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8006,ERR2146929,ERX2203462,ERS1952820,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D5,SAMEA104327842,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327842|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D5 p,XY D5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D5_R1.fastq.gz XY_D5_R2.fastq.gz,fastq fastq,660547188.0,2621219.0,E MTAB 6077:XY D5 R,0:126 1:126,A:188043609;C:145685742;G:136282398;T:190482268;N:53171,126,126,,,188043609,145685742,136282398,190482268,53171,ERX2203462,ERS1952820,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81152,0.81307,0.24567,0.24771,0.9152,0.9162,0.62955,0.42909,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8007,ERR2146928,ERX2203461,ERS1952819,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D4,SAMEA104327841,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327841|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D4 p,XY D4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D4_R1.fastq.gz XY_D4_R2.fastq.gz,fastq fastq,837525276.0,3323513.0,E MTAB 6077:XY D4 R,0:126 1:126,A:236023482;C:186075815;G:177194660;T:238169798;N:61521,126,126,,,236023482,186075815,177194660,238169798,61521,ERX2203461,ERS1952819,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.89213,0.89192,0.12147,0.12558,0.9083,0.91062,0.51202,0.51681,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8008,ERR2146927,ERX2203460,ERS1952818,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D3,SAMEA104327840,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327840|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D3 p,XY D3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D3_R1.fastq.gz XY_D3_R2.fastq.gz,fastq fastq,929369952.0,3687976.0,E MTAB 6077:XY D3 R,0:126 1:126,A:268574709;C:200604190;G:190405734;T:269719266;N:66053,126,126,,,268574709,200604190,190405734,269719266,66053,ERX2203460,ERS1952818,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85669,0.85766,0.26039,0.26737,0.91948,0.92113,0.52044,0.51927,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8009,ERR2146926,ERX2203459,ERS1952817,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D2,SAMEA104327839,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327839|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D2 p,XY D2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D2_R1.fastq.gz XY_D2_R2.fastq.gz,fastq fastq,689447808.0,2735904.0,E MTAB 6077:XY D2 R,0:126 1:126,A:196263920;C:152901761;G:142462888;T:197767203;N:52036,126,126,,,196263920,152901761,142462888,197767203,52036,ERX2203459,ERS1952817,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81038,0.81259,0.1659,0.16893,0.91587,0.91758,0.6298,0.6291,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8010,ERR2146925,ERX2203458,ERS1952816,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D1,SAMEA104327838,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327838|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D1 p,XY D1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D1_R1.fastq.gz XY_D1_R2.fastq.gz,fastq fastq,1074802428.0,4265089.0,E MTAB 6077:XY D1 R,0:126 1:126,A:295122432;C:249510143;G:233653745;T:296436723;N:79385,126,126,,,295122432,249510143,233653745,296436723,79385,ERX2203458,ERS1952816,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.88767,0.88989,0.14758,0.15226,0.90096,0.90254,0.50951,0.50804,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8011,ERR2146924,ERX2203457,ERS1952815,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C5,SAMEA104327837,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327837|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C5 p,XY C5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C5_R1.fastq.gz XY_C5_R2.fastq.gz,fastq fastq,763663068.0,3030409.0,E MTAB 6077:XY C5 R,0:126 1:126,A:218783973;C:168189496;G:157662131;T:218961836;N:65632,126,126,,,218783973,168189496,157662131,218961836,65632,ERX2203457,ERS1952815,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.78078,0.78189,0.22088,0.22216,0.92429,0.92476,0.6094,0.60799,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8012,ERR2146923,ERX2203456,ERS1952814,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C4,SAMEA104327836,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327836|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C4 p,XY C4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C4_R1.fastq.gz XY_C4_R2.fastq.gz,fastq fastq,628535880.0,2494190.0,E MTAB 6077:XY C4 R,0:126 1:126,A:178148646;C:141113691;G:131448439;T:177778949;N:46155,126,126,,,178148646,141113691,131448439,177778949,46155,ERX2203456,ERS1952814,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.73091,0.73106,0.16098,0.16176,0.9303,0.93048,0.75666,0.75542,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8013,ERR2146922,ERX2203455,ERS1952813,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C3,SAMEA104327835,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327835|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C3 p,XY C3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C3_R2.fastq.gz XY_C3_R1.fastq.gz,fastq fastq,1143928548.0,4539399.0,E MTAB 6077:XY C3 R,0:126 1:126,A:333818058;C:244426168;G:233177976;T:332420812;N:85534,126,126,,,333818058,244426168,233177976,332420812,85534,ERX2203455,ERS1952813,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85792,0.85722,0.28359,0.28517,0.91725,0.91703,0.49423,0.49677,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8014,ERR2146921,ERX2203454,ERS1952812,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C2,SAMEA104327834,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327834|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C2 p,XY C2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C2_R1.fastq.gz XY_C2_R2.fastq.gz,fastq fastq,1109822868.0,4404059.0,E MTAB 6077:XY C2 R,0:126 1:126,A:315349917;C:244964515;G:234207255;T:315223120;N:78061,126,126,,,315349917,244964515,234207255,315223120,78061,ERX2203454,ERS1952812,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.89018,0.88918,0.18419,0.18554,0.90617,0.90698,0.51664,0.5151,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8015,ERR2146920,ERX2203453,ERS1952811,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C1,SAMEA104327833,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327833|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C1 p,XY C1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C1_R1.fastq.gz XY_C1_R2.fastq.gz,fastq fastq,1023038100.0,4059675.0,E MTAB 6077:XY C1 R,0:126 1:126,A:289423372;C:226901377;G:218604509;T:288035093;N:73749,126,126,,,289423372,226901377,218604509,288035093,73749,ERX2203453,ERS1952811,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.8847,0.88446,0.09904,0.10044,0.91187,0.91273,0.50218,0.50146,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8016,ERR2146919,ERX2203452,ERS1952810,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B5,SAMEA104327832,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327832|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B5 p,XY B5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B5_R1.fastq.gz XY_B5_R2.fastq.gz,fastq fastq,1157792580.0,4594415.0,E MTAB 6077:XY B5 R,0:126 1:126,A:332377265;C:250239737;G:240957301;T:334125637;N:92640,126,126,,,332377265,250239737,240957301,334125637,92640,ERX2203452,ERS1952810,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.88876,0.88961,0.2351,0.23604,0.89509,0.89485,0.43495,0.50926,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8017,ERR2146918,ERX2203451,ERS1952809,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B4,SAMEA104327831,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327831|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B4 p,XY B4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B4_R1.fastq.gz XY_B4_R2.fastq.gz,fastq fastq,1210007988.0,4801619.0,E MTAB 6077:XY B4 R,0:126 1:126,A:347394878;C:262886787;G:250224660;T:349411084;N:90579,126,126,,,347394878,262886787,250224660,349411084,90579,ERX2203451,ERS1952809,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.88955,0.88989,0.24237,0.24323,0.90794,0.90735,0.49869,0.49848,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8018,ERR2146917,ERX2203450,ERS1952808,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B3,SAMEA104327830,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327830|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B3 p,XY B3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B3_R1.fastq.gz XY_B3_R2.fastq.gz,fastq fastq,1176133896.0,4667198.0,E MTAB 6077:XY B3 R,0:126 1:126,A:335919936;C:258107071;G:247773671;T:334248508;N:84710,126,126,,,335919936,258107071,247773671,334248508,84710,ERX2203450,ERS1952808,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.84653,0.84743,0.19732,0.19994,0.92178,0.92186,0.51636,0.48599,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8019,ERR2146916,ERX2203449,ERS1952807,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B2,SAMEA104327829,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327829|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B2 p,XY B2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B2_R1.fastq.gz XY_B2_R2.fastq.gz,fastq fastq,690569208.0,2740354.0,E MTAB 6077:XY B2 R,0:126 1:126,A:198979532;C:149863253;G:142526360;T:199148360;N:51703,126,126,,,198979532,149863253,142526360,199148360,51703,ERX2203449,ERS1952807,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80432,0.80459,0.18079,0.18089,0.92368,0.92395,0.66657,0.66039,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8020,ERR2146915,ERX2203448,ERS1952806,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B1,SAMEA104327828,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327828|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B1 p,XY B1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B1_R1.fastq.gz XY_B1_R2.fastq.gz,fastq fastq,1099919772.0,4364761.0,E MTAB 6077:XY B1 R,0:126 1:126,A:320782014;C:232447890;G:224643364;T:321968049;N:78455,126,126,,,320782014,232447890,224643364,321968049,78455,ERX2203448,ERS1952806,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.86247,0.863,0.30591,0.30783,0.90155,0.90199,0.52033,0.52673,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8021,ERR2146914,ERX2203447,ERS1952805,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A6,SAMEA104327827,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327827|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:21Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A6|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A6|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A6 p,XY A6 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A6|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A6_R1.fastq.gz XY_A6_R2.fastq.gz,fastq fastq,512695008.0,2034504.0,E MTAB 6077:XY A6 R,0:126 1:126,A:142817402;C:116839066;G:108882918;T:144112206;N:43416,126,126,,,142817402,116839066,108882918,144112206,43416,ERX2203447,ERS1952805,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.68622,0.68692,0.14751,0.14781,0.93825,0.93906,0.74578,0.74484,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8022,ERR2146913,ERX2203446,ERS1952804,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A5,SAMEA104327826,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327826|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A5|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A5|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A5 p,XY A5 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A5|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A5_R1.fastq.gz XY_A5_R2.fastq.gz,fastq fastq,781978176.0,3103088.0,E MTAB 6077:XY A5 R,0:126 1:126,A:213601286;C:183203675;G:166424027;T:218688044;N:61144,126,126,,,213601286,183203675,166424027,218688044,61144,ERX2203446,ERS1952804,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.70128,0.70185,0.23891,0.24003,0.92316,0.92305,0.5932,0.58961,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8023,ERR2146912,ERX2203445,ERS1952803,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A4,SAMEA104327825,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327825|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A4|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A4|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A4 p,XY A4 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A4|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A4_R1.fastq.gz XY_A4_R2.fastq.gz,fastq fastq,963604152.0,3823826.0,E MTAB 6077:XY A4 R,0:126 1:126,A:274833754;C:210148026;G:199842855;T:278711035;N:68482,126,126,,,274833754,210148026,199842855,278711035,68482,ERX2203445,ERS1952803,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85087,0.85235,0.23408,0.23608,0.90278,0.90423,0.52332,0.51671,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8024,ERR2146911,ERX2203444,ERS1952802,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A3,SAMEA104327824,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327824|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A3|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A3|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A3 p,XY A3 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A3|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A3_R1.fastq.gz XY_A3_R2.fastq.gz,fastq fastq,659648052.0,2617651.0,E MTAB 6077:XY A3 R,0:126 1:126,A:189357994;C:143277472;G:136135683;T:190829498;N:47405,126,126,,,189357994,143277472,136135683,190829498,47405,ERX2203444,ERS1952802,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.79506,0.79671,0.18692,0.18896,0.92529,0.92644,0.60886,0.60533,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8025,ERR2146910,ERX2203443,ERS1952801,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A2,SAMEA104327823,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327823|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A2|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A2|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A2 p,XY A2 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A2|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A2_R1.fastq.gz XY_A2_R2.fastq.gz,fastq fastq,1128768228.0,4479239.0,E MTAB 6077:XY A2 R,0:126 1:126,A:308213498;C:261633998;G:246008259;T:312829796;N:82677,126,126,,,308213498,261633998,246008259,312829796,82677,ERX2203443,ERS1952801,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87413,0.87359,0.16944,0.17295,0.89631,0.89741,0.5001,0.49423,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8026,ERR2146909,ERX2203442,ERS1952800,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A1,SAMEA104327822,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327822|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A1|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A1|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A1 p,XY A1 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A1|Experimental Factor: block:batch 2 2015 04 08,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A1_R1.fastq.gz XY_A1_R2.fastq.gz,fastq fastq,788118660.0,3127455.0,E MTAB 6077:XY A1 R,0:126 1:126,A:224956393;C:172218956;G:163283909;T:227604645;N:54757,126,126,,,224956393,172218956,163283909,227604645,54757,ERX2203442,ERS1952800,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80149,0.80336,0.21497,0.21728,0.91088,0.91161,0.5548,0.55294,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8027,ERR2146908,ERX2203441,ERS1952799,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H9,SAMEA104327821,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327821|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H9 p,XY H9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H9|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H9_R1.fastq.gz XY_H9_R2.fastq.gz,fastq fastq,807594228.0,3204739.0,E MTAB 6077:XY H9 R,0:126 1:126,A:218595449;C:187636861;G:181518262;T:219784313;N:59343,126,126,,,218595449,187636861,181518262,219784313,59343,ERX2203441,ERS1952799,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.78544,0.78636,0.05597,0.05666,0.93269,0.93221,0.43413,0.42718,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8028,ERR2146907,ERX2203440,ERS1952798,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H11,SAMEA104327820,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327820|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H11|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H11|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H11 p,XY H11 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H11|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H11_R1.fastq.gz XY_H11_R2.fastq.gz,fastq fastq,1192565052.0,4732401.0,E MTAB 6077:XY H11 R,0:126 1:126,A:342932563;C:257052157;G:248657956;T:343834739;N:87637,126,126,,,342932563,257052157,248657956,343834739,87637,ERX2203440,ERS1952798,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.89616,0.89684,0.16772,0.16999,0.89658,0.89702,0.52116,0.52033,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8029,ERR2146906,ERX2203439,ERS1952797,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY H10,SAMEA104327819,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327819|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY H10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY H10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY H10 p,XY H10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY H10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_H10_R1.fastq.gz XY_H10_R2.fastq.gz,fastq fastq,657055980.0,2607365.0,E MTAB 6077:XY H10 R,0:126 1:126,A:185904003;C:143873783;G:140155273;T:187074871;N:48050,126,126,,,185904003,143873783,140155273,187074871,48050,ERX2203439,ERS1952797,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87268,0.87184,0.20288,0.20565,0.89158,0.89175,0.50978,0.51761,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8030,ERR2146905,ERX2203438,ERS1952796,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G9,SAMEA104327818,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327818|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G9 p,XY G9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G9|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G9_R1.fastq.gz XY_G9_R2.fastq.gz,fastq fastq,848036196.0,3365223.0,E MTAB 6077:XY G9 R,0:126 1:126,A:226227423;C:202347981;G:190590823;T:228811695;N:58274,126,126,,,226227423,202347981,190590823,228811695,58274,ERX2203438,ERS1952796,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.67126,0.66849,0.04553,0.04585,0.95268,0.95329,0.49116,0.49344,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8031,ERR2146904,ERX2203437,ERS1952795,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G11,SAMEA104327817,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327817|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G11|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G11|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G11 p,XY G11 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G11|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G11_R1.fastq.gz XY_G11_R2.fastq.gz,fastq fastq,1195583256.0,4744378.0,E MTAB 6077:XY G11 R,0:126 1:126,A:345424111;C:261338773;G:243193915;T:345540435;N:86022,126,126,,,345424111,261338773,243193915,345540435,86022,ERX2203437,ERS1952795,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.76911,0.77056,0.24714,0.24897,0.94728,0.94769,0.66489,0.66853,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8032,ERR2146903,ERX2203436,ERS1952794,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G10,SAMEA104327816,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327816|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY G10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G10 p,XY G10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G10_R1.fastq.gz XY_G10_R2.fastq.gz,fastq fastq,639397332.0,2537291.0,E MTAB 6077:XY G10 R,0:126 1:126,A:173456517;C:148128754;G:143479252;T:174284508;N:48301,126,126,,,173456517,148128754,143479252,174284508,48301,ERX2203436,ERS1952794,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80875,0.80581,0.0537,0.05433,0.94286,0.94249,0.4914,0.49421,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8033,ERR2146902,ERX2203435,ERS1952793,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F9,SAMEA104327815,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327815|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F9 p,XY F9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F9|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F9_R1.fastq.gz XY_F9_R2.fastq.gz,fastq fastq,1006950168.0,3995834.0,E MTAB 6077:XY F9 R,0:126 1:126,A:283197386;C:223742397;G:213220755;T:286719135;N:70495,126,126,,,283197386,223742397,213220755,286719135,70495,ERX2203435,ERS1952793,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.8812,0.88049,0.1659,0.16779,0.90076,0.90096,0.49325,0.48816,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8034,ERR2146901,ERX2203434,ERS1952792,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F11,SAMEA104327814,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327814|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F11|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F11|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F11 p,XY F11 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F11|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F11_R1.fastq.gz XY_F11_R2.fastq.gz,fastq fastq,1202087628.0,4770189.0,E MTAB 6077:XY F11 R,0:126 1:126,A:349592736;C:259170175;G:244309124;T:348928664;N:86929,126,126,,,349592736,259170175,244309124,348928664,86929,ERX2203434,ERS1952792,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87213,0.87252,0.18705,0.18823,0.90015,0.90074,0.4557,0.53699,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8035,ERR2146900,ERX2203433,ERS1952791,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY F10,SAMEA104327813,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327813|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY F10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY F10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY F10 p,XY F10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY F10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_F10_R1.fastq.gz XY_F10_R2.fastq.gz,fastq fastq,747528768.0,2966384.0,E MTAB 6077:XY F10 R,0:126 1:126,A:210091874;C:165907726;G:159579247;T:211898681;N:51240,126,126,,,210091874,165907726,159579247,211898681,51240,ERX2203433,ERS1952791,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80413,0.80426,0.22448,0.22632,0.93322,0.93346,0.48943,0.49505,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8036,ERR2146899,ERX2203432,ERS1952790,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E11,SAMEA104327812,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327812|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E11|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E11|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E11 p,XY E11 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E11|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E11_R1.fastq.gz XY_E11_R2.fastq.gz,fastq fastq,1150342956.0,4564853.0,E MTAB 6077:XY E11 R,0:126 1:126,A:325273328;C:259272677;G:241496334;T:324217090;N:83527,126,126,,,325273328,259272677,241496334,324217090,83527,ERX2203432,ERS1952790,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.76178,0.76406,0.08061,0.08102,0.93606,0.93592,0.52694,0.53148,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8037,ERR2146898,ERX2203431,ERS1952789,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E10,SAMEA104327811,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327811|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY E10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E10 p,XY E10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E10_R2.fastq.gz XY_E10_R1.fastq.gz,fastq fastq,683322192.0,2711596.0,E MTAB 6077:XY E10 R,0:126 1:126,A:188884574;C:155711125;G:147372810;T:191302114;N:51569,126,126,,,188884574,155711125,147372810,191302114,51569,ERX2203431,ERS1952789,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.66721,0.67032,0.16164,0.16383,0.92823,0.92872,0.5223,0.51809,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8038,ERR2146897,ERX2203430,ERS1952788,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D9,SAMEA104327810,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327810|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D9 p,XY D9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D9|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D9_R1.fastq.gz XY_D9_R2.fastq.gz,fastq fastq,865979100.0,3436425.0,E MTAB 6077:XY D9 R,0:126 1:126,A:240616655;C:197599165;G:183681566;T:244020738;N:60976,126,126,,,240616655,197599165,183681566,244020738,60976,ERX2203430,ERS1952788,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.74906,0.75308,0.16198,0.1657,0.92342,0.92468,0.52614,0.51676,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8039,ERR2146896,ERX2203429,ERS1952787,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D12,SAMEA104327809,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327809|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D12|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D12|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D12 p,XY D12 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D12|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D12_R1.fastq.gz XY_D12_R2.fastq.gz,fastq fastq,905176944.0,3591972.0,E MTAB 6077:XY D12 R,0:126 1:126,A:246555073;C:216983812;G:200573378;T:240986928;N:77753,126,126,,,246555073,216983812,200573378,240986928,77753,ERX2203429,ERS1952787,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.85062,0.85094,0.11736,0.11979,0.91327,0.91386,0.47263,0.46533,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8040,ERR2146895,ERX2203428,ERS1952786,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D11,SAMEA104327808,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327808|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D11|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D11|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D11 p,XY D11 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D11|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D11_R1.fastq.gz XY_D11_R2.fastq.gz,fastq fastq,1291953348.0,5126799.0,E MTAB 6077:XY D11 R,0:126 1:126,A:369920538;C:287114506;G:267740879;T:367079058;N:98367,126,126,,,369920538,287114506,267740879,367079058,98367,ERX2203428,ERS1952786,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.87096,0.87237,0.24196,0.2461,0.90792,0.90899,0.52339,0.5271,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8041,ERR2146894,ERX2203427,ERS1952785,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY D10,SAMEA104327807,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327807|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY D10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY D10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY D10 p,XY D10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY D10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_D10_R1.fastq.gz XY_D10_R2.fastq.gz,fastq fastq,748715184.0,2971092.0,E MTAB 6077:XY D10 R,0:126 1:126,A:208061705;C:169498579;G:160126085;T:210976858;N:51957,126,126,,,208061705,169498579,160126085,210976858,51957,ERX2203427,ERS1952785,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.66206,0.66846,0.17043,0.17467,0.94485,0.94537,0.59533,0.59546,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8042,ERR2146893,ERX2203426,ERS1952784,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C9,SAMEA104327806,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327806|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C9 p,XY C9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C9|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C9_R1.fastq.gz XY_C9_R2.fastq.gz,fastq fastq,1150429392.0,4565196.0,E MTAB 6077:XY C9 R,0:126 1:126,A:326536641;C:254922524;G:241800832;T:327087874;N:81521,126,126,,,326536641,254922524,241800832,327087874,81521,ERX2203426,ERS1952784,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.86595,0.86669,0.20417,0.20552,0.90477,0.90477,0.54058,0.55202,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8043,ERR2146892,ERX2203425,ERS1952783,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C12,SAMEA104327805,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327805|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C12|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C12|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C12 p,XY C12 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C12|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C12_R1.fastq.gz XY_C12_R2.fastq.gz,fastq fastq,831898116.0,3301183.0,E MTAB 6077:XY C12 R,0:126 1:126,A:227429111;C:198577064;G:187365045;T:218459452;N:67444,126,126,,,227429111,198577064,187365045,218459452,67444,ERX2203425,ERS1952783,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.83467,0.83397,0.12537,0.1255,0.91634,0.9161,0.46295,0.4679,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8044,ERR2146891,ERX2203424,ERS1952782,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY C10,SAMEA104327804,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327804|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY C10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY C10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY C10 p,XY C10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY C10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_C10_R1.fastq.gz XY_C10_R2.fastq.gz,fastq fastq,759926412.0,3015581.0,E MTAB 6077:XY C10 R,0:126 1:126,A:218046053;C:164343382;G:157901837;T:219577671;N:57469,126,126,,,218046053,164343382,157901837,219577671,57469,ERX2203424,ERS1952782,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.81097,0.81114,0.17208,0.1725,0.91192,0.91271,0.5607,0.55845,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8045,ERR2146890,ERX2203423,ERS1952781,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B9,SAMEA104327803,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327803|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B9 p,XY B9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B9|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B9_R1.fastq.gz XY_B9_R2.fastq.gz,fastq fastq,1013795496.0,4022998.0,E MTAB 6077:XY B9 R,0:126 1:126,A:286799345;C:224069384;G:215079127;T:287772660;N:74980,126,126,,,286799345,224069384,215079127,287772660,74980,ERX2203423,ERS1952781,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.84668,0.84801,0.13945,0.14132,0.90345,0.90343,0.49384,0.48897,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8046,ERR2146889,ERX2203422,ERS1952780,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B11,SAMEA104327802,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327802|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B11|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B11|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B11 p,XY B11 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B11|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B11_R1.fastq.gz XY_B11_R2.fastq.gz,fastq fastq,1456956648.0,5781574.0,E MTAB 6077:XY B11 R,0:126 1:126,A:420203574;C:317178129;G:303061432;T:416404384;N:109129,126,126,,,420203574,317178129,303061432,416404384,109129,ERX2203422,ERS1952780,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.86953,0.87066,0.177,0.17864,0.91344,0.91376,0.45985,0.457,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8047,ERR2146888,ERX2203421,ERS1952779,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY B10,SAMEA104327801,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327801|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY B10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY B10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY B10 p,XY B10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY B10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_B10_R1.fastq.gz XY_B10_R2.fastq.gz,fastq fastq,593982144.0,2357072.0,E MTAB 6077:XY B10 R,0:126 1:126,A:171955349;C:126578476;G:122662307;T:172744277;N:41735,126,126,,,171955349,126578476,122662307,172744277,41735,ERX2203421,ERS1952779,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.82753,0.8298,0.19732,0.19989,0.90496,0.90676,0.54908,0.55062,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8048,ERR2146887,ERX2203420,ERS1952778,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A12,SAMEA104327800,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327800|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A12|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A12|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A12 p,XY A12 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A12|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A12_R1.fastq.gz XY_A12_R2.fastq.gz,fastq fastq,1230682824.0,4883662.0,E MTAB 6077:XY A12 R,0:126 1:126,A:355010960;C:266762902;G:251920887;T:356886941;N:101134,126,126,,,355010960,266762902,251920887,356886941,101134,ERX2203420,ERS1952778,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.86322,0.86375,0.29406,0.29552,0.91153,0.91261,0.57808,0.58221,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8049,ERR2146886,ERX2203419,ERS1952777,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A11,SAMEA104327799,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327799|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A11|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A11|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A11 p,XY A11 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A11|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A11_R1.fastq.gz XY_A11_R2.fastq.gz,fastq fastq,1296460620.0,5144685.0,E MTAB 6077:XY A11 R,0:126 1:126,A:355230694;C:308719376;G:280706624;T:351711962;N:91964,126,126,,,355230694,308719376,280706624,351711962,91964,ERX2203419,ERS1952777,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.76747,0.7683,0.15226,0.1536,0.9207,0.92082,0.53597,0.44233,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8050,ERR2146885,ERX2203418,ERS1952776,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY A10,SAMEA104327798,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327798|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY A10|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:pass|sample name:E MTAB 6077:XY A10|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY A10 p,XY A10 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY A10|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_A10_R1.fastq.gz XY_A10_R2.fastq.gz,fastq fastq,671732712.0,2665606.0,E MTAB 6077:XY A10 R,0:126 1:126,A:185165370;C:152925443;G:147448536;T:186145700;N:47663,126,126,,,185165370,152925443,147448536,186145700,47663,ERX2203418,ERS1952776,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.80656,0.80709,0.1229,0.12496,0.9153,0.91642,0.55337,0.54811,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8051,ERR2146884,ERX2203417,ERS1952775,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY G8,SAMEA104327797,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327797|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:20Z|INSDC status:public|Submitter Id:E MTAB 6077:XY G8|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:fail|sample name:E MTAB 6077:XY G8|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY G8 p,XY G8 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY G8|Experimental Factor: block:batch 3 2015 04 15,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_G8_R1.fastq.gz XY_G8_R2.fastq.gz,fastq fastq,588343896.0,2334698.0,E MTAB 6077:XY G8 R,0:126 1:126,A:161664342;C:137911851;G:127926021;T:160790247;N:51435,126,126,,,161664342,137911851,127926021,160790247,51435,ERX2203417,ERS1952775,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.59013,0.59239,0.06895,0.06948,0.9684,0.96895,0.70036,0.68614,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures
8052,ERR2146883,ERX2203416,ERS1952774,ERP104575,PRJEB22861,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E-MTAB-6077,Transcriptome Analysis,Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer 10.7554/eLife.03848. Specifically  the 2.7 kb Mouse Smarcd3 F6 sequence chr5: 24113559  24116342 from mm9 assembly was sub cloned from a gateway entry vector into the Zebrafish Enhancer Detection ZED. Tol2 mediated transgenesis was performed and stable lines were created. The Smarcd3 F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3 F6:EGFPhsc70 embryos were dissociated at 10 hpf for fluorescent activated cell sorting FACS to collect GFP+ cells. Single cell cDNA libraries were prepared using Fluidgim C1 system Fluidgim  PN 100 7168 Rev. B1. 96 single cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells number of genes detected > 2000 were kept for clustering analysis. For the tube control experiments we performed mRNA seq on 4000 cells from the same embryo batches as the scRNA seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP  comparison.  Libraries were made following the Fluidgim C1 mRNA seq tube control protocol Fluidgim  PN 100 7168 Rev. B1. Polyadenylated mRNA was captured by Oligo dT primers and PCR amplified post reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair end sequenced on the Illumina HiSeq 2500 platform.,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2017 10 03,,Protocols: Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature. Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction. Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,XY E9,SAMEA104327796,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto",ENA first public:2018 08 20|ENA last update:2017 10 03|External Id:SAMEA104327796|INSDC center alias:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC center name:Genetics and Genome Biology Program  Hospital for Sick Children  Toronto  Canada; Department of Molecular Genetics  University of Toronto|INSDC first public:2018 08 20T17:01:55Z|INSDC last update:2017 10 03T09:08:19Z|INSDC status:public|Submitter Id:E MTAB 6077:XY E9|age:10|broker name:ArrayExpress|common name:zebrafish|developmental stage:gastrula bud|genotype:Smarcd3 F6:EGFPhsc70|organism part:whole organism|phenotype:GFP postitive|post analysis well quantity:fail|sample name:E MTAB 6077:XY E9|well information:single cell,,,,,,,,,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,E MTAB 6077:XY E9 p,XY E9 p,Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,Around 100 TgSmarcd3 F6: EGFP embryos were dechorionated with pronase Sigma  Cat# 11459643001 and transferred to a 1.5mL Eppendorf tube when they reached bud stage. post incubated in 200ul calcium free Ringer solution 116 mM NaCl  2.6mM KCl  5mM HEPE  pH 7.0 for 5 min  embryos were transferred into a 24 well plate filled with 500 ul TrypLE solution GIBOCO  TrypLE Express Enzyme  cat #: 12604 013 for dissociation at room temperature.  Embryos were gently homogenized every 5min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in single cell suspension. The cell suspension was transferred into 200ul ice cold FBS to stop the reaction. Cells were centrifuged at 300g for 3min at 4 degree and washed with 500 ul ice cold DMEM with 10% FBS before resuspended in 500 ul ice cold DMEM with 1% FBS. Right before the FACS  cells were filtered through a 40um strainer and DAPI was added at a concentration of 5 ug/ml to exclude dead cells. FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter with a 100um nozzle by the SickKids UHN Flow and Mass Cytometry Facility. Cell doublets and dead cells were excluded based on forward scatter  side scatter and DAPI channel. GFP+ and GFP  cells were sorted into 100% FBS and subjected to RNA seq or procedures immediately once sorting finished. 30 000 50 000 GFP+ cells and 100  000 GFP  cells were usually collected in one sort. Single cell cDNA libraries were prepared using Fluidgim C1 system. post FACS  GFP positive cells were washed twice in DMEM with 3%FBS and filtered through a 40um cell strainer. Cells were adjusted to a concentration of 400 500 cells/ul before mixed with C1 suspension solution at a 5.2:4.8 ratio. Then 10ul final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. Reverse transcription  PCR amplification and cDNA harvesting were performed as per manufactures instruction.  Since the optimized capture efficiency of C1 chip for our cell is 30 40%  96 single cell cDNA libraries were collected from three batches of experiments at the end. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit Illumina  Cat# FC 131 1096.,Experimental Factor: phenotype:GFP postitive|Experimental Factor: single cell identifier:XY E9|Experimental Factor: block:batch 1 2015 03 25,RNA-Seq,TRANSCRIPTOMIC,Oligo-dT,PAIRED,ILLUMINA,Illumina HiSeq 2500,<SPOT_DESCRIPTOR><SPOT_DECODE_SPEC><SPOT_LENGTH>252</SPOT_LENGTH><READ_SPEC><READ_INDEX>0</READ_INDEX><READ_LABEL>F</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Forward</READ_TYPE><BASE_COORD>1</BASE_COORD></READ_SPEC><READ_SPEC><READ_INDEX>1</READ_INDEX><READ_LABEL>R</READ_LABEL><READ_CLASS>Application Read</READ_CLASS><READ_TYPE>Reverse</READ_TYPE><BASE_COORD>127</BASE_COORD></READ_SPEC></SPOT_DECODE_SPEC></SPOT_DESCRIPTOR>,ERP104575,Illumina HiSeq 2500 paired end sequencing; Determining single cell mRNA expression profiles of a specific population isolated from zebrafish embryos at 10 hpf,ENA FIRST PUBLIC:2018 08 20|ENA LAST UPDATE:2018 11 16,XY_E9_R1.fastq.gz XY_E9_R2.fastq.gz,fastq fastq,757841364.0,3007307.0,E MTAB 6077:XY E9 R,0:126 1:126,A:209040696;C:173808092;G:163540899;T:211398494;N:53183,126,126,,,209040696,173808092,163540899,211398494,53183,ERX2203416,ERS1952774,ERA1108167,"Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive","Genetics and Genome Biology Program, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto|European Nucleotide Archive",2,0.60954,0.61079,0.26381,0.26464,0.96063,0.96124,0.71926,0.72414,126,126,B,B,biological fallback assumption,illumina,hiseq_era,unknown,poly_a,nextera,sc_generic,single_cell_generic,generic-scrnaseq-only,,Canada,2017-10-03,Gastrula,Embryo,Whole Organism,All anatomical structures