{"database": "metadata", "table": "run_metadata", "rows": [[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", null, "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", null, null, null, null, null, null, null, null, "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", "2520FApplication ReadForward11RApplication ReadReverse127", "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, null, null, 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", null, "Canada", "2017-10-03", "Gastrula", "Embryo", "Whole Organism", "All anatomical structures"]], "columns": ["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"], "primary_keys": ["rowid"], "primary_key_values": ["8023"], "units": {}, "query_ms": 10.13198000146076}