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
47758,SRR6890914,SRX3841335,SRS3087495,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo tyrosinase C,GSM3067206,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,CRISPR Targeted Embryo tyrosinase C,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,GSM3067206,GSM3067206: CRISPR Targeted Embryo tyrosinase C; Danio rerio; RNA Seq,GSM3067206,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067206,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW203.fastq.gz,fastq,1465038283.0,25330646.0,GSM3067206 r1,0:57.84,A:399530311;C:292071189;G:290193165;T:483241417;N:2201,57,,,,399530311,292071189,290193165,483241417,2201,SRX3841335,SRS3087495,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88338,,0.13639,,0.78295,,0.48646,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47759,SRR6890915,SRX3841335,SRS3087495,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo tyrosinase C,GSM3067206,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,CRISPR Targeted Embryo tyrosinase C,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,GSM3067206,GSM3067206: CRISPR Targeted Embryo tyrosinase C; Danio rerio; RNA Seq,GSM3067206,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067206,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW204.fastq.gz,fastq,1462027529.0,25327645.0,GSM3067206 r2,0:57.72,A:396943012;C:291806975;G:292100327;T:481174977;N:2238,57,,,,396943012,291806975,292100327,481174977,2238,SRX3841335,SRS3087495,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88375,,0.13748,,0.78589,,0.49104,,60,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47760,SRR6890916,SRX3841335,SRS3087495,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo tyrosinase C,GSM3067206,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,CRISPR Targeted Embryo tyrosinase C,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,GSM3067206,GSM3067206: CRISPR Targeted Embryo tyrosinase C; Danio rerio; RNA Seq,GSM3067206,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067206,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW205.fastq.gz,fastq,1089879965.0,19024780.0,GSM3067206 r3,0:57.29,A:299577336;C:213749796;G:214826324;T:361724880;N:1629,57,,,,299577336,213749796,214826324,361724880,1629,SRX3841335,SRS3087495,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88076,,0.13757,,0.78324,,0.4895,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47761,SRR6890912,SRX3841334,SRS3087494,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo tyrosinase B,GSM3067205,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,CRISPR Targeted Embryo tyrosinase B,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,GSM3067205,GSM3067205: CRISPR Targeted Embryo tyrosinase B; Danio rerio; RNA Seq,GSM3067205,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067205,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW198.fastq.gz,fastq,1750603340.0,30262674.0,GSM3067205 r1,0:57.85,A:476772061;C:348089294;G:341349666;T:584389613;N:2706,57,,,,476772061,348089294,341349666,584389613,2706,SRX3841334,SRS3087494,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88041,,0.13852,,0.79052,,0.50356,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47762,SRR6890913,SRX3841334,SRS3087494,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo tyrosinase B,GSM3067205,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,CRISPR Targeted Embryo tyrosinase B,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,GSM3067205,GSM3067205: CRISPR Targeted Embryo tyrosinase B; Danio rerio; RNA Seq,GSM3067205,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067205,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW199.fastq.gz,fastq,1719747339.0,29740919.0,GSM3067205 r2,0:57.82,A:465181744;C:338602328;G:336843629;T:579117056;N:2582,57,,,,465181744,338602328,336843629,579117056,2582,SRX3841334,SRS3087494,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.89022,,0.13973,,0.79046,,0.47161,,60,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47763,SRR6890910,SRX3841333,SRS3087493,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo tyrosinase A,GSM3067204,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,CRISPR Targeted Embryo tyrosinase A,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,GSM3067204,GSM3067204: CRISPR Targeted Embryo tyrosinase A; Danio rerio; RNA Seq,GSM3067204,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067204,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW191.fastq.gz,fastq,1430918940.0,24757119.0,GSM3067204 r1,0:57.80,A:386848505;C:283215419;G:279611431;T:481241360;N:2225,57,,,,386848505,283215419,279611431,481241360,2225,SRX3841333,SRS3087493,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88097,,0.1317,,0.79722,,0.50104,,20,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47764,SRR6890911,SRX3841333,SRS3087493,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo tyrosinase A,GSM3067204,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,CRISPR Targeted Embryo tyrosinase A,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:tyrosinase targeted Cas9,GSM3067204,GSM3067204: CRISPR Targeted Embryo tyrosinase A; Danio rerio; RNA Seq,GSM3067204,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067204,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW192.fastq.gz,fastq,1460183677.0,25279012.0,GSM3067204 r2,0:57.76,A:393214592;C:288843309;G:287072676;T:491050801;N:2299,57,,,,393214592,288843309,287072676,491050801,2299,SRX3841333,SRS3087493,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88188,,0.13315,,0.79878,,0.47807,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47765,SRR6890907,SRX3841332,SRS3087492,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin C,GSM3067203,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin C,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067203,GSM3067203: CRISPR Targeted Embryo chordin C; Danio rerio; RNA Seq,GSM3067203,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067203,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW206.fastq.gz,fastq,1176220272.0,20354110.0,GSM3067203 r1,0:57.79,A:321130163;C:233656608;G:233960194;T:387471545;N:1762,57,,,,321130163,233656608,233960194,387471545,1762,SRX3841332,SRS3087492,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88335,,0.15046,,0.78559,,0.52197,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47766,SRR6890908,SRX3841332,SRS3087492,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin C,GSM3067203,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin C,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067203,GSM3067203: CRISPR Targeted Embryo chordin C; Danio rerio; RNA Seq,GSM3067203,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067203,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW207.fastq.gz,fastq,1094042604.0,18956943.0,GSM3067203 r2,0:57.71,A:299520746;C:217679611;G:217383022;T:359457558;N:1667,57,,,,299520746,217679611,217383022,359457558,1667,SRX3841332,SRS3087492,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87731,,0.14126,,0.78455,,0.51361,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47767,SRR6890909,SRX3841332,SRS3087492,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin C,GSM3067203,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin C,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067203,GSM3067203: CRISPR Targeted Embryo chordin C; Danio rerio; RNA Seq,GSM3067203,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067203,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW208.fastq.gz,fastq,1096168422.0,18997947.0,GSM3067203 r3,0:57.70,A:299037227;C:219614732;G:219564739;T:357950083;N:1641,57,,,,299037227,219614732,219564739,357950083,1641,SRX3841332,SRS3087492,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87903,,0.1438,,0.78652,,0.50674,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47768,SRR6890904,SRX3841331,SRS3087491,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin B,GSM3067202,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin B,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067202,GSM3067202: CRISPR Targeted Embryo chordin B; Danio rerio; RNA Seq,GSM3067202,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067202,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW200.fastq.gz,fastq,1515827613.0,26281497.0,GSM3067202 r1,0:57.68,A:416122854;C:299949702;G:299356908;T:500395913;N:2236,57,,,,416122854,299949702,299356908,500395913,2236,SRX3841331,SRS3087491,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87584,,0.15434,,0.78522,,0.51754,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47769,SRR6890905,SRX3841331,SRS3087491,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin B,GSM3067202,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin B,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067202,GSM3067202: CRISPR Targeted Embryo chordin B; Danio rerio; RNA Seq,GSM3067202,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067202,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW201.fastq.gz,fastq,1486290397.0,25698034.0,GSM3067202 r2,0:57.84,A:405024414;C:294656621;G:294652070;T:491954998;N:2294,57,,,,405024414,294656621,294652070,491954998,2294,SRX3841331,SRS3087491,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88539,,0.1652,,0.78922,,0.51537,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47770,SRR6890906,SRX3841331,SRS3087491,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin B,GSM3067202,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin B,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067202,GSM3067202: CRISPR Targeted Embryo chordin B; Danio rerio; RNA Seq,GSM3067202,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067202,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW202.fastq.gz,fastq,1381539779.0,23983713.0,GSM3067202 r3,0:57.60,A:376598971;C:275317367;G:275659505;T:453961857;N:2079,57,,,,376598971,275317367,275659505,453961857,2079,SRX3841331,SRS3087491,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88032,,0.15709,,0.79017,,0.5072,,58,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47771,SRR6890899,SRX3841330,SRS3087490,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin A,GSM3067201,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin A,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067201,GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq,GSM3067201,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067201,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW193.fastq.gz,fastq,1319530737.0,22907290.0,GSM3067201 r1,0:57.60,A:357858623;C:262292188;G:266165318;T:433212743;N:1865,57,,,,357858623,262292188,266165318,433212743,1865,SRX3841330,SRS3087490,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.8749,,0.13612,,0.79691,,0.51509,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47772,SRR6890900,SRX3841330,SRS3087490,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin A,GSM3067201,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin A,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067201,GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq,GSM3067201,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067201,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW194.fastq.gz,fastq,1291827031.0,22431130.0,GSM3067201 r2,0:57.59,A:350651901;C:256281684;G:263900265;T:420991184;N:1997,57,,,,350651901,256281684,263900265,420991184,1997,SRX3841330,SRS3087490,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87725,,0.13374,,0.79651,,0.53079,,41,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47773,SRR6890901,SRX3841330,SRS3087490,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin A,GSM3067201,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin A,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067201,GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq,GSM3067201,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067201,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW195.fastq.gz,fastq,1227344744.0,21231899.0,GSM3067201 r3,0:57.81,A:334536880;C:242949864;G:245894782;T:403961308;N:1910,57,,,,334536880,242949864,245894782,403961308,1910,SRX3841330,SRS3087490,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87642,,0.13817,,0.79397,,0.52786,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47774,SRR6890902,SRX3841330,SRS3087490,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin A,GSM3067201,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin A,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067201,GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq,GSM3067201,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067201,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW196.fastq.gz,fastq,1311849779.0,22690047.0,GSM3067201 r4,0:57.82,A:356392127;C:259146968;G:263404525;T:432904086;N:2073,57,,,,356392127,259146968,263404525,432904086,2073,SRX3841330,SRS3087490,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88119,,0.13756,,0.79409,,0.52718,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47775,SRR6890903,SRX3841330,SRS3087490,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,CRISPR Targeted Embryo chordin A,GSM3067201,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,CRISPR Targeted Embryo chordin A,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:14hpf|treatment:embryos injected with sgRNA and Cas9 protein at xxx cell stage|genotype:chordin targeted Cas9,GSM3067201,GSM3067201: CRISPR Targeted Embryo chordin A; Danio rerio; RNA Seq,GSM3067201,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067201,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW197.fastq.gz,fastq,1346161233.0,23336788.0,GSM3067201 r5,0:57.68,A:366046254;C:265311327;G:272129691;T:442671952;N:2009,57,,,,366046254,265311327,272129691,442671952,2009,SRX3841330,SRS3087490,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87736,,0.13479,,0.79255,,0.53336,,60,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47776,SRR6890896,SRX3841329,SRS3087489,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 5,GSM3067200,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 5,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067200,GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq,GSM3067200,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067200,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW167.fastq.gz,fastq,811220746.0,14099347.0,GSM3067200 r1,0:57.54,A:226775040;C:163877565;G:173170450;T:247395403;N:2288,57,,,,226775040,163877565,173170450,247395403,2288,SRX3841329,SRS3087489,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88233,,0.12759,,0.79082,,0.51035,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47777,SRR6890897,SRX3841329,SRS3087489,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 5,GSM3067200,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 5,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067200,GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq,GSM3067200,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067200,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW168.fastq.gz,fastq,824982674.0,14331143.0,GSM3067200 r2,0:57.57,A:231177547;C:167432521;G:176426897;T:249943460;N:2249,57,,,,231177547,167432521,176426897,249943460,2249,SRX3841329,SRS3087489,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88583,,0.12411,,0.79277,,0.52031,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47778,SRR6890898,SRX3841329,SRS3087489,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 5,GSM3067200,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 5,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067200,GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq,GSM3067200,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067200,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW169.fastq.gz,fastq,693186309.0,12065222.0,GSM3067200 r3,0:57.45,A:197516210;C:138205483;G:144601216;T:212861628;N:1772,57,,,,197516210,138205483,144601216,212861628,1772,SRX3841329,SRS3087489,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.86621,,0.12722,,0.78823,,0.51859,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47779,SRR11699740,SRX3841329,SRS3087489,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 5,GSM3067200,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 5,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067200,GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq,GSM3067200,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067200,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW167.gfp.fastq.gz,fastq,637401493.0,11247658.0,GSM3067200 r11,0:56.67,A:211763567;C:130934906;G:157778746;T:136920350;N:3924,56,,,,211763567,130934906,157778746,136920350,3924,SRX3841329,SRS3087489,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00818,,0.0001,,0.99902,,0.79816,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47780,SRR11699741,SRX3841329,SRS3087489,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 5,GSM3067200,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 5,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067200,GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq,GSM3067200,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067200,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW168.gfp.fastq.gz,fastq,648934432.0,11464121.0,GSM3067200 r12,0:56.61,A:211915696;C:134836103;G:161336635;T:140841785;N:4213,56,,,,211915696,134836103,161336635,140841785,4213,SRX3841329,SRS3087489,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.0103,,0.00018,,0.99853,,0.78199,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47781,SRR11699742,SRX3841329,SRS3087489,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 5,GSM3067200,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 5,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067200,GSM3067200: TracerSeq Embryo 5; Danio rerio; RNA Seq,GSM3067200,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067200,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW169.gfp.fastq.gz,fastq,555982541.0,9807444.0,GSM3067200 r13,0:56.69,A:182949392;C:115632932;G:137856329;T:119540249;N:3639,56,,,,182949392,115632932,137856329,119540249,3639,SRX3841329,SRS3087489,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.01235,,0.00032,,0.99868,,0.81431,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47782,SRR6890891,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW162.fastq.gz,fastq,884280521.0,15352773.0,GSM3067199 r1,0:57.60,A:248995548;C:176713956;G:184425201;T:274143441;N:2375,57,,,,248995548,176713956,184425201,274143441,2375,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87729,,0.1278,,0.77904,,0.51481,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47783,SRR6890892,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW163.fastq.gz,fastq,952171001.0,16579450.0,GSM3067199 r2,0:57.43,A:269068875;C:192967485;G:199258236;T:290873928;N:2477,57,,,,269068875,192967485,199258236,290873928,2477,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.86963,,0.12299,,0.78261,,0.51621,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47784,SRR6890893,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW164.fastq.gz,fastq,1133026695.0,19698928.0,GSM3067199 r3,0:57.52,A:320544827;C:227286162;G:235085716;T:350106942;N:3048,57,,,,320544827,227286162,235085716,350106942,3048,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87678,,0.12527,,0.78169,,0.50623,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47785,SRR6890894,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW165.fastq.gz,fastq,793581204.0,13818281.0,GSM3067199 r4,0:57.43,A:223975396;C:158365631;G:165125978;T:246112109;N:2090,57,,,,223975396,158365631,165125978,246112109,2090,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87803,,0.12874,,0.78374,,0.50983,,44,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47786,SRR6890895,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW166.fastq.gz,fastq,792728001.0,13800321.0,GSM3067199 r5,0:57.44,A:225238995;C:161675115;G:167314732;T:238497072;N:2087,57,,,,225238995,161675115,167314732,238497072,2087,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87776,,0.11531,,0.79458,,0.53005,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47787,SRR11699735,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW162.gfp.fastq.gz,fastq,725258705.0,12804354.0,GSM3067199 r11,0:56.64,A:244821707;C:144850405;G:178149916;T:157432024;N:4653,56,,,,244821707,144850405,178149916,157432024,4653,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00953,,0.00045,,0.99874,,0.84025,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47788,SRR11699736,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW163.gfp.fastq.gz,fastq,709146800.0,12584798.0,GSM3067199 r12,0:56.35,A:239870182;C:143213119;G:172165989;T:153892922;N:4588,56,,,,239870182,143213119,172165989,153892922,4588,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00541,,0.00022,,0.99888,,0.84231,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47789,SRR11699737,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW164.gfp.fastq.gz,fastq,648001757.0,11476883.0,GSM3067199 r13,0:56.46,A:220229172;C:129764679;G:155857424;T:142146491;N:3991,56,,,,220229172,129764679,155857424,142146491,3991,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00638,,0.00028,,0.999,,0.82363,,53,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47790,SRR11699738,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW165.gfp.fastq.gz,fastq,596792506.0,10531209.0,GSM3067199 r14,0:56.67,A:200618246;C:122094142;G:144891967;T:129184490;N:3661,56,,,,200618246,122094142,144891967,129184490,3661,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00789,,0.00046,,0.99888,,0.84071,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47791,SRR11699739,SRX3841328,SRS3087488,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 4,GSM3067199,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 4,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067199,GSM3067199: TracerSeq Embryo 4; Danio rerio; RNA Seq,GSM3067199,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067199,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW166.gfp.fastq.gz,fastq,566854985.0,10065909.0,GSM3067199 r15,0:56.31,A:192383179;C:111486159;G:139172733;T:123809364;N:3550,56,,,,192383179,111486159,139172733,123809364,3550,SRX3841328,SRS3087488,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00671,,0.00038,,0.99886,,0.85738,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47792,SRR6890887,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW158.fastq.gz,fastq,965976032.0,16779564.0,GSM3067198 r1,0:57.57,A:270793370;C:196343924;G:207973938;T:290862136;N:2664,57,,,,270793370,196343924,207973938,290862136,2664,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88031,,0.11243,,0.7919,,0.51366,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47793,SRR6890888,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW159.fastq.gz,fastq,1015306225.0,17632016.0,GSM3067198 r2,0:57.58,A:282554762;C:206663548;G:220061729;T:306023342;N:2844,57,,,,282554762,206663548,220061729,306023342,2844,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88781,,0.11491,,0.79251,,0.51135,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47794,SRR6890889,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW160.fastq.gz,fastq,958415543.0,16704238.0,GSM3067198 r3,0:57.38,A:267638469;C:191936701;G:204743895;T:294094008;N:2470,57,,,,267638469,191936701,204743895,294094008,2470,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87973,,0.12017,,0.78922,,0.48734,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47795,SRR6890890,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW161.fastq.gz,fastq,911486560.0,15853985.0,GSM3067198 r4,0:57.49,A:254067794;C:182351617;G:196560408;T:278504296;N:2445,57,,,,254067794,182351617,196560408,278504296,2445,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88691,,0.12363,,0.78435,,0.50693,,60,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47796,SRR11699731,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW158.gfp.fastq.gz,fastq,714356770.0,12690001.0,GSM3067198 r11,0:56.29,A:238801241;C:146515701;G:173416188;T:155618953;N:4687,56,,,,238801241,146515701,173416188,155618953,4687,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00355,,0.00025,,0.99902,,0.70542,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47797,SRR11699732,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW159.gfp.fastq.gz,fastq,692190367.0,12308538.0,GSM3067198 r12,0:56.24,A:231865817;C:139854334;G:169105319;T:151360408;N:4489,56,,,,231865817,139854334,169105319,151360408,4489,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00277,,0.00018,,0.99931,,0.70414,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47798,SRR11699733,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW160.gfp.fastq.gz,fastq,713049432.0,12694194.0,GSM3067198 r13,0:56.17,A:239502412;C:145346774;G:172186875;T:156008904;N:4467,56,,,,239502412,145346774,172186875,156008904,4467,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00331,,0.00023,,0.9991,,0.70743,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47799,SRR11699734,SRX3841327,SRS3087487,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 3,GSM3067198,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 3,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067198,GSM3067198: TracerSeq Embryo 3; Danio rerio; RNA Seq,GSM3067198,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067198,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW161.gfp.fastq.gz,fastq,753741455.0,13357439.0,GSM3067198 r14,0:56.43,A:251557444;C:154340741;G:181807551;T:166030917;N:4802,56,,,,251557444,154340741,181807551,166030917,4802,SRX3841327,SRS3087487,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.0034,,0.00026,,0.99924,,0.74673,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47800,SRR6890881,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW105.fastq.gz,fastq,2236392658.0,38613668.0,GSM3067197 r1,0:57.92,A:632525539;C:430906734;G:458150451;T:714800928;N:9006,57,,,,632525539,430906734,458150451,714800928,9006,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88225,,0.11769,,0.77895,,0.49448,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47801,SRR6890882,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW106.fastq.gz,fastq,2231823587.0,38523508.0,GSM3067197 r2,0:57.93,A:630838902;C:434227222;G:457362069;T:709386360;N:9034,57,,,,630838902,434227222,457362069,709386360,9034,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88583,,0.11501,,0.78171,,0.51228,,60,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47802,SRR6890883,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW107.fastq.gz,fastq,2017895917.0,34862336.0,GSM3067197 r3,0:57.88,A:571926612;C:389743099;G:413210412;T:643007658;N:8136,57,,,,571926612,389743099,413210412,643007658,8136,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88214,,0.11984,,0.78234,,0.5107,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47803,SRR6890884,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW108.fastq.gz,fastq,2137179290.0,36984331.0,GSM3067197 r4,,,,,,,,,,,,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",,,,,,,,,,,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47804,SRR6890885,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW109.fastq.gz,fastq,1732824461.0,29964040.0,GSM3067197 r5,0:57.83,A:488970094;C:337910710;G:363385259;T:542551597;N:6801,57,,,,488970094,337910710,363385259,542551597,6801,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88484,,0.11274,,0.78782,,0.52053,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47805,SRR6890886,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW110.fastq.gz,fastq,1869184778.0,32270092.0,GSM3067197 r6,0:57.92,A:523455278;C:363490974;G:393498418;T:588732594;N:7514,57,,,,523455278,363490974,393498418,588732594,7514,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.89075,,0.11527,,0.78435,,0.516,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47806,SRR11699725,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW105.gfp.fastq.gz,fastq,864508154.0,15268405.0,GSM3067197 r11,0:56.62,A:280405131;C:177118247;G:209076103;T:197907076;N:1597,56,,,,280405131,177118247,209076103,197907076,1597,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00246,,0.00014,,0.99945,,0.57366,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47807,SRR11699726,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW106.gfp.fastq.gz,fastq,1836795274.0,32436891.0,GSM3067197 r12,0:56.63,A:598413910;C:375443322;G:442230080;T:420704508;N:3454,56,,,,598413910,375443322,442230080,420704508,3454,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00339,,0.00022,,0.99904,,0.81609,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47808,SRR11699727,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW107.gfp.fastq.gz,fastq,1098090093.0,19584542.0,GSM3067197 r13,0:56.07,A:358936332;C:225531082;G:265165807;T:248454887;N:1985,56,,,,358936332,225531082,265165807,248454887,1985,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00394,,0.00027,,0.99924,,0.79207,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47809,SRR11699728,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW108.gfp.fastq.gz,fastq,973690024.0,17208640.0,GSM3067197 r14,0:56.58,A:315592395;C:202218220;G:234572367;T:221305217;N:1825,56,,,,315592395,202218220,234572367,221305217,1825,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00289,,0.00018,,0.99943,,0.77946,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47810,SRR11699729,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW109.gfp.fastq.gz,fastq,860964317.0,15239252.0,GSM3067197 r15,0:56.50,A:280154617;C:178242732;G:206646971;T:195918375;N:1622,56,,,,280154617,178242732,206646971,195918375,1622,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.0029,,0.00016,,0.9992,,0.8034,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47811,SRR11699730,SRX3841326,SRS3087486,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 2,GSM3067197,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 2,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067197,GSM3067197: TracerSeq Embryo 2; Danio rerio; RNA Seq,GSM3067197,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067197,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW110.gfp.fastq.gz,fastq,353310699.0,6684362.0,GSM3067197 r16,0:52.86,A:116163854;C:71721845;G:85203701;T:80220874;N:425,52,,,,116163854,71721845,85203701,80220874,425,SRX3841326,SRS3087486,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00187,,0.00013,,0.99959,,0.84967,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47812,SRR6890877,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW101.fastq.gz,fastq,2986488041.0,51575075.0,GSM3067196 r1,0:57.91,A:848651747;C:586829293;G:609184887;T:941810264;N:11850,57,,,,848651747,586829293,609184887,941810264,11850,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88677,,0.11046,,0.7905,,0.53466,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47813,SRR6890878,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW102.fastq.gz,fastq,2367681335.0,40881016.0,GSM3067196 r2,0:57.92,A:671355626;C:465619829;G:484744401;T:745951960;N:9519,57,,,,671355626,465619829,484744401,745951960,9519,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.8812,,0.10473,,0.78884,,0.51963,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47814,SRR6890879,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW103.fastq.gz,fastq,2331578063.0,40348694.0,GSM3067196 r3,0:57.79,A:661550584;C:462384104;G:479423343;T:728210706;N:9326,57,,,,661550584,462384104,479423343,728210706,9326,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88193,,0.10461,,0.78839,,0.52362,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47815,SRR6890880,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW104.fastq.gz,fastq,2049500633.0,35395333.0,GSM3067196 r4,0:57.90,A:579583630;C:404370534;G:423144188;T:642394171;N:8110,57,,,,579583630,404370534,423144188,642394171,8110,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88736,,0.10152,,0.78727,,0.49826,,61,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47816,SRR11699721,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW101.gfp.fastq.gz,fastq,770401542.0,13588300.0,GSM3067196 r11,0:56.70,A:252451711;C:161581601;G:188319243;T:168047500;N:1487,56,,,,252451711,161581601,188319243,168047500,1487,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00572,,0.00021,,0.99912,,0.78671,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47817,SRR11699722,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW102.gfp.fastq.gz,fastq,930082189.0,16450240.0,GSM3067196 r12,0:56.54,A:307009540;C:193290503;G:225634898;T:204145591;N:1657,56,,,,307009540,193290503,225634898,204145591,1657,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00499,,0.00023,,0.99908,,0.82519,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47818,SRR11699723,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW103.gfp.fastq.gz,fastq,1127167312.0,19917784.0,GSM3067196 r13,0:56.59,A:373217299;C:236443835;G:273368582;T:244135449;N:2147,56,,,,373217299,236443835,273368582,244135449,2147,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.00429,,0.00014,,0.99922,,0.82,,60,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47819,SRR11699724,SRX3841325,SRS3087485,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,TracerSeq Embryo 1,GSM3067196,,tissue:Zebrafish Embryo Dissociated Cells|strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,TracerSeq Embryo 1,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:AB|developmental stage:24hpf|treatment:embryos injected with TracerSeq library and Tol2 transposase mRNA at xxx cell stage|genotype:wild type,GSM3067196,GSM3067196: TracerSeq Embryo 1; Danio rerio; RNA Seq,GSM3067196,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067196,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName,DEW104.gfp.fastq.gz,fastq,975575896.0,17282185.0,GSM3067196 r14,0:56.45,A:321010798;C:205573092;G:237075384;T:211914717;N:1905,56,,,,321010798,205573092,237075384,211914717,1905,SRX3841325,SRS3087485,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.0035,,8e-05,,0.99924,,0.83208,,61,,T,,under 1.2% mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47820,SRR6890870,SRX3841324,SRS3087484,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  24hpf,GSM3067195,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  24hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,GSM3067195,GSM3067195: Zebrafish Embryos  24hpf; Danio rerio; RNA Seq,GSM3067195,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067195,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW021.fastq.gz,fastq,2019482704.0,60636489.0,GSM3067195 r1,0:33.30,A:493417632;C:419033159;G:432871824;T:674158736;N:1353,33,,,,493417632,419033159,432871824,674158736,1353,SRX3841324,SRS3087484,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.82488,,0.11082,,0.78522,,0.51753,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47821,SRR6890871,SRX3841324,SRS3087484,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  24hpf,GSM3067195,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  24hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,GSM3067195,GSM3067195: Zebrafish Embryos  24hpf; Danio rerio; RNA Seq,GSM3067195,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067195,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW052.fastq.gz,fastq,1757215612.0,52765096.0,GSM3067195 r2,0:33.30,A:441169837;C:344094059;G:363706881;T:608242069;N:2766,33,,,,441169837,344094059,363706881,608242069,2766,SRX3841324,SRS3087484,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.83362,,0.12368,,0.78553,,0.52928,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47822,SRR6890872,SRX3841324,SRS3087484,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  24hpf,GSM3067195,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  24hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,GSM3067195,GSM3067195: Zebrafish Embryos  24hpf; Danio rerio; RNA Seq,GSM3067195,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067195,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW053.fastq.gz,fastq,1821864281.0,54670237.0,GSM3067195 r3,,,,,,,,,,,,SRX3841324,SRS3087484,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.82644,,0.12187,,0.78784,,0.52385,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47823,SRR6890873,SRX3841324,SRS3087484,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  24hpf,GSM3067195,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  24hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,GSM3067195,GSM3067195: Zebrafish Embryos  24hpf; Danio rerio; RNA Seq,GSM3067195,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067195,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW054.fastq.gz,fastq,1678567945.0,50389624.0,GSM3067195 r4,0:33.31,A:409485468;C:338736084;G:352566121;T:577777533;N:2739,33,,,,409485468,338736084,352566121,577777533,2739,SRX3841324,SRS3087484,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.84082,,0.12109,,0.78744,,0.5204,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47824,SRR6890874,SRX3841324,SRS3087484,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  24hpf,GSM3067195,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  24hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,GSM3067195,GSM3067195: Zebrafish Embryos  24hpf; Danio rerio; RNA Seq,GSM3067195,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067195,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW055.fastq.gz,fastq,1767167982.0,53040354.0,GSM3067195 r5,0:33.32,A:434649073;C:352847211;G:367753051;T:611915672;N:2975,33,,,,434649073,352847211,367753051,611915672,2975,SRX3841324,SRS3087484,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.83659,,0.12547,,0.78626,,0.51721,,31,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47825,SRR6890875,SRX3841324,SRS3087484,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  24hpf,GSM3067195,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  24hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,GSM3067195,GSM3067195: Zebrafish Embryos  24hpf; Danio rerio; RNA Seq,GSM3067195,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067195,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW056.fastq.gz,fastq,1848811670.0,55500253.0,GSM3067195 r6,0:33.31,A:454979688;C:370746083;G:384432449;T:638650390;N:3060,33,,,,454979688,370746083,384432449,638650390,3060,SRX3841324,SRS3087484,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.83588,,0.12238,,0.78723,,0.51497,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47826,SRR6890876,SRX3841324,SRS3087484,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  24hpf,GSM3067195,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  24hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:24hpf|treatment:untreated embryos|genotype:wild type,GSM3067195,GSM3067195: Zebrafish Embryos  24hpf; Danio rerio; RNA Seq,GSM3067195,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067195,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW057.fastq.gz,fastq,1748505537.0,52490191.0,GSM3067195 r7,0:33.31,A:445047455;C:338487369;G:357051616;T:607916307;N:2790,33,,,,445047455,338487369,357051616,607916307,2790,SRX3841324,SRS3087484,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.82436,,0.11709,,0.79192,,0.50453,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Pharyngula,Embryo,Embryo Imprecise,All anatomical structures
47827,SRR6890863,SRX3841323,SRS3087483,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  18hpf,GSM3067194,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  18hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,GSM3067194,GSM3067194: Zebrafish Embryos  18hpf; Danio rerio; RNA Seq,GSM3067194,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067194,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW001.fastq.gz,fastq,5964444855.0,154387997.0,GSM3067194 r1,0:38.63,A:1472956070;C:1228455711;G:1338025807;T:1924940927;N:66340,38,,,,1472956070,1228455711,1338025807,1924940927,66340,SRX3841323,SRS3087483,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.90322,,0.12802,,0.78449,,0.5166,,32,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47828,SRR6890864,SRX3841323,SRS3087483,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  18hpf,GSM3067194,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  18hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,GSM3067194,GSM3067194: Zebrafish Embryos  18hpf; Danio rerio; RNA Seq,GSM3067194,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067194,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW003.fastq.gz,fastq,10762044173.0,282099745.0,GSM3067194 r2,0:38.15,A:2863738417;C:2156355507;G:2317056975;T:3424778770;N:114504,38,,,,2863738417,2156355507,2317056975,3424778770,114504,SRX3841323,SRS3087483,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.87127,,0.08766,,0.79833,,0.51556,,29,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47829,SRR6890865,SRX3841323,SRS3087483,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  18hpf,GSM3067194,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  18hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,GSM3067194,GSM3067194: Zebrafish Embryos  18hpf; Danio rerio; RNA Seq,GSM3067194,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067194,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW012.fastq.gz,fastq,2160282606.0,60764540.0,GSM3067194 r3,0:35.55,A:540267067;C:424959324;G:475172425;T:719883014;N:776,35,,,,540267067,424959324,475172425,719883014,776,SRX3841323,SRS3087483,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.89224,,0.11121,,0.78681,,0.49218,,16,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47830,SRR6890866,SRX3841323,SRS3087483,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  18hpf,GSM3067194,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  18hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,GSM3067194,GSM3067194: Zebrafish Embryos  18hpf; Danio rerio; RNA Seq,GSM3067194,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067194,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW038.fastq.gz,fastq,2501392133.0,74952238.0,GSM3067194 r4,0:33.37,A:588874023;C:505543466;G:528398121;T:878574824;N:1699,33,,,,588874023,505543466,528398121,878574824,1699,SRX3841323,SRS3087483,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85776,,0.1437,,0.78287,,0.51179,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47831,SRR6890867,SRX3841323,SRS3087483,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  18hpf,GSM3067194,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  18hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,GSM3067194,GSM3067194: Zebrafish Embryos  18hpf; Danio rerio; RNA Seq,GSM3067194,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067194,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW039.fastq.gz,fastq,2590149863.0,77584377.0,GSM3067194 r5,0:33.38,A:606121706;C:527691833;G:552446644;T:903887707;N:1973,33,,,,606121706,527691833,552446644,903887707,1973,SRX3841323,SRS3087483,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.8556,,0.14432,,0.78466,,0.52334,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47832,SRR6890868,SRX3841323,SRS3087483,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  18hpf,GSM3067194,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  18hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,GSM3067194,GSM3067194: Zebrafish Embryos  18hpf; Danio rerio; RNA Seq,GSM3067194,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067194,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW040.fastq.gz,fastq,1355757822.0,40622007.0,GSM3067194 r6,0:33.37,A:317774317;C:272430840;G:291175247;T:474376465;N:953,33,,,,317774317,272430840,291175247,474376465,953,SRX3841323,SRS3087483,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85879,,0.14444,,0.78052,,0.51168,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47833,SRR6890869,SRX3841323,SRS3087483,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  18hpf,GSM3067194,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  18hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:18hpf|treatment:untreated embryos|genotype:wild type,GSM3067194,GSM3067194: Zebrafish Embryos  18hpf; Danio rerio; RNA Seq,GSM3067194,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067194,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW041.fastq.gz,fastq,2377609297.0,71217226.0,GSM3067194 r7,0:33.39,A:557520223;C:479488603;G:506743618;T:833855136;N:1717,33,,,,557520223,479488603,506743618,833855136,1717,SRX3841323,SRS3087483,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.8596,,0.14974,,0.77869,,0.50763,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47834,SRR6890859,SRX3841322,SRS3087482,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  14hpf,GSM3067193,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  14hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,GSM3067193,GSM3067193: Zebrafish Embryos  14hpf; Danio rerio; RNA Seq,GSM3067193,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067193,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW011.fastq.gz,fastq,1783957341.0,50224064.0,GSM3067193 r1,0:35.52,A:446335050;C:346598253;G:387688911;T:603334543;N:584,35,,,,446335050,346598253,387688911,603334543,584,SRX3841322,SRS3087482,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.89079,,0.12469,,0.79137,,0.48967,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47835,SRR6890860,SRX3841322,SRS3087482,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  14hpf,GSM3067193,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  14hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,GSM3067193,GSM3067193: Zebrafish Embryos  14hpf; Danio rerio; RNA Seq,GSM3067193,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067193,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW035.fastq.gz,fastq,2511325160.0,75228701.0,GSM3067193 r2,0:33.38,A:595180535;C:506267676;G:529233170;T:880641896;N:1883,33,,,,595180535,506267676,529233170,880641896,1883,SRX3841322,SRS3087482,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85303,,0.1467,,0.79198,,0.5082,,23,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47836,SRR6890861,SRX3841322,SRS3087482,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  14hpf,GSM3067193,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  14hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,GSM3067193,GSM3067193: Zebrafish Embryos  14hpf; Danio rerio; RNA Seq,GSM3067193,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067193,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW036.fastq.gz,fastq,2549967719.0,76368280.0,GSM3067193 r3,0:33.39,A:593754908;C:518473002;G:538552208;T:899185771;N:1830,33,,,,593754908,518473002,538552208,899185771,1830,SRX3841322,SRS3087482,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85633,,0.14916,,0.79385,,0.52614,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47837,SRR6890862,SRX3841322,SRS3087482,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  14hpf,GSM3067193,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  14hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:14hpf|treatment:untreated embryos|genotype:wild type,GSM3067193,GSM3067193: Zebrafish Embryos  14hpf; Danio rerio; RNA Seq,GSM3067193,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067193,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW037.fastq.gz,fastq,2477774899.0,74187211.0,GSM3067193 r4,0:33.40,A:578365312;C:501416585;G:519884899;T:878106212;N:1891,33,,,,578365312,501416585,519884899,878106212,1891,SRX3841322,SRS3087482,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85414,,0.15107,,0.79202,,0.52285,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Segmentation,Embryo,Embryo Imprecise,All anatomical structures
47838,SRR6890855,SRX3841321,SRS3087481,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  10hpf,GSM3067192,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  10hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,GSM3067192,GSM3067192: Zebrafish Embryos  10hpf; Danio rerio; RNA Seq,GSM3067192,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067192,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW010.fastq.gz,fastq,2110862021.0,59408212.0,GSM3067192 r1,0:35.53,A:527302725;C:408047237;G:458389764;T:717121584;N:711,35,,,,527302725,408047237,458389764,717121584,711,SRX3841321,SRS3087481,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.88725,,0.12485,,0.79916,,0.50249,,35,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47839,SRR6890856,SRX3841321,SRS3087481,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  10hpf,GSM3067192,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  10hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,GSM3067192,GSM3067192: Zebrafish Embryos  10hpf; Danio rerio; RNA Seq,GSM3067192,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067192,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW032.fastq.gz,fastq,1690310046.0,50614131.0,GSM3067192 r2,0:33.40,A:392480786;C:340035561;G:356671545;T:601120887;N:1267,33,,,,392480786,340035561,356671545,601120887,1267,SRX3841321,SRS3087481,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85214,,0.13084,,0.80387,,0.51566,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47840,SRR6890857,SRX3841321,SRS3087481,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  10hpf,GSM3067192,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  10hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,GSM3067192,GSM3067192: Zebrafish Embryos  10hpf; Danio rerio; RNA Seq,GSM3067192,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067192,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW033.fastq.gz,fastq,1715874376.0,51389870.0,GSM3067192 r3,0:33.39,A:402622060;C:341051995;G:362643647;T:609555459;N:1215,33,,,,402622060,341051995,362643647,609555459,1215,SRX3841321,SRS3087481,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85353,,0.13375,,0.8029,,0.52106,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47841,SRR6890858,SRX3841321,SRS3087481,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  10hpf,GSM3067192,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  10hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:10hpf|treatment:untreated embryos|genotype:wild type,GSM3067192,GSM3067192: Zebrafish Embryos  10hpf; Danio rerio; RNA Seq,GSM3067192,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067192,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW034.fastq.gz,fastq,1723859055.0,51625754.0,GSM3067192 r4,0:33.39,A:402083188;C:343192837;G:366312678;T:612269130;N:1222,33,,,,402083188,343192837,366312678,612269130,1222,SRX3841321,SRS3087481,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.85321,,0.13421,,0.80426,,0.52915,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47842,SRR6890851,SRX3841320,SRS3087480,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  8hpf,GSM3067191,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  8hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,GSM3067191,GSM3067191: Zebrafish Embryos  8hpf; Danio rerio; RNA Seq,GSM3067191,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067191,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW046.fastq.gz,fastq,1702483582.0,51072575.0,GSM3067191 r1,0:33.33,A:419022097;C:323609574;G:337964317;T:621884823;N:2771,33,,,,419022097,323609574,337964317,621884823,2771,SRX3841320,SRS3087480,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.8324,,0.17303,,0.81801,,0.54866,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47843,SRR6890852,SRX3841320,SRS3087480,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  8hpf,GSM3067191,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  8hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,GSM3067191,GSM3067191: Zebrafish Embryos  8hpf; Danio rerio; RNA Seq,GSM3067191,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067191,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW047.fastq.gz,fastq,1788403190.0,53653761.0,GSM3067191 r2,0:33.33,A:448532000;C:330949061;G:348064862;T:660854395;N:2872,33,,,,448532000,330949061,348064862,660854395,2872,SRX3841320,SRS3087480,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.82969,,0.16961,,0.81832,,0.5461,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47844,SRR6890853,SRX3841320,SRS3087480,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  8hpf,GSM3067191,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  8hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,GSM3067191,GSM3067191: Zebrafish Embryos  8hpf; Danio rerio; RNA Seq,GSM3067191,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067191,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW048.fastq.gz,fastq,1723652489.0,51707851.0,GSM3067191 r3,0:33.33,A:431207220;C:320830082;G:340076335;T:631536024;N:2828,33,,,,431207220,320830082,340076335,631536024,2828,SRX3841320,SRS3087480,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.82925,,0.17248,,0.81803,,0.5504,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47845,SRR6890854,SRX3841320,SRS3087480,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  8hpf,GSM3067191,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  8hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:08hpf|treatment:untreated embryos|genotype:wild type,GSM3067191,GSM3067191: Zebrafish Embryos  8hpf; Danio rerio; RNA Seq,GSM3067191,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067191,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW049.fastq.gz,fastq,1689373817.0,50673079.0,GSM3067191 r4,0:33.34,A:413427546;C:320409992;G:334119220;T:621414280;N:2779,33,,,,413427546,320409992,334119220,621414280,2779,SRX3841320,SRS3087480,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.83512,,0.16614,,0.81903,,0.54045,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47846,SRR6890847,SRX3841319,SRS3087479,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  6hpf,GSM3067190,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  6hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,GSM3067190,GSM3067190: Zebrafish Embryos  6hpf; Danio rerio; RNA Seq,GSM3067190,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067190,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW042.fastq.gz,fastq,1568138624.0,47031488.0,GSM3067190 r1,0:33.34,A:383645031;C:295130721;G:309738139;T:579622263;N:2470,33,,,,383645031,295130721,309738139,579622263,2470,SRX3841319,SRS3087479,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.83316,,0.14754,,0.80628,,0.55449,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47847,SRR6890848,SRX3841319,SRS3087479,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  6hpf,GSM3067190,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  6hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,GSM3067190,GSM3067190: Zebrafish Embryos  6hpf; Danio rerio; RNA Seq,GSM3067190,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067190,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW043.fastq.gz,fastq,1813251144.0,54390933.0,GSM3067190 r2,0:33.34,A:452137395;C:336059336;G:357138872;T:667912590;N:2951,33,,,,452137395,336059336,357138872,667912590,2951,SRX3841319,SRS3087479,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.83173,,0.14839,,0.80874,,0.54878,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47848,SRR6890849,SRX3841319,SRS3087479,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  6hpf,GSM3067190,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  6hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,GSM3067190,GSM3067190: Zebrafish Embryos  6hpf; Danio rerio; RNA Seq,GSM3067190,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067190,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW044.fastq.gz,fastq,1562214573.0,46906051.0,GSM3067190 r3,0:33.31,A:387099457;C:290021123;G:306440621;T:578650950;N:2422,33,,,,387099457,290021123,306440621,578650950,2422,SRX3841319,SRS3087479,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.8317,,0.14852,,0.80693,,0.54447,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47849,SRR6890850,SRX3841319,SRS3087479,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  6hpf,GSM3067190,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  6hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:06hpf|treatment:untreated embryos|genotype:wild type,GSM3067190,GSM3067190: Zebrafish Embryos  6hpf; Danio rerio; RNA Seq,GSM3067190,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067190,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW045.fastq.gz,fastq,2495609024.0,74858057.0,GSM3067190 r4,0:33.34,A:622971438;C:464022853;G:480774815;T:927835940;N:3978,33,,,,622971438,464022853,480774815,927835940,3978,SRX3841319,SRS3087479,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.82948,,0.1439,,0.81132,,0.55832,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Gastrula,Embryo,Embryo Imprecise,All anatomical structures
47850,SRR6890845,SRX3841318,SRS3087478,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  4hpf,GSM3067189,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:04hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  4hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:04hpf|treatment:untreated embryos|genotype:wild type,GSM3067189,GSM3067189: Zebrafish Embryos  4hpf; Danio rerio; RNA Seq,GSM3067189,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067189,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW050.fastq.gz,fastq,2094113704.0,62868338.0,GSM3067189 r1,0:33.31,A:527011614;C:422792400;G:431192702;T:713113521;N:3467,33,,,,527011614,422792400,431192702,713113521,3467,SRX3841318,SRS3087478,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.84951,,0.13721,,0.78397,,0.63113,,33,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Blastula,Embryo,Embryo Imprecise,All anatomical structures
47851,SRR6890846,SRX3841318,SRS3087478,SRP136369,PRJNA445487,Systematic mapping of cell state trajectories  cell lineage  and perturbations in the zebrafish embryo using single cell transcriptomics,GSE112294,Transcriptome Analysis,High throughput mapping of cellular differentiation hierarchies from single cell data promises to empower systematic interrogations of vertebrate development and disease.  Here  we applied single cell RNA sequencing to >92 000 cells from zebrafish embryos during the first day of development. Using a graph based approach  we mapped a cell state landscape that describes axis patterning  germ layer formation  and organogenesis.  We tested how clonally related cells traverse this landscape by developing a transposon based barcoding approach “TracerSeq” for reconstructing single cell lineage histories. Clonally related cells were often restricted by the state landscape  including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in chordin deficient embryos. We provide web based resources for further analysis of the single cell data. Overall design: Single cell mRNA sequencing of zebrafish embryonic cells. Samples1 7: Single cell libraries from untreated embryos 4 hpf 24 hpf Samples8 12: Single cell libraries from embryos injected with TracerSeq lineage cassette at the 1 cell stage. Samples13 18: Single cell libraries from embryos injected with sgRNA + Cas9 at the 1 cell stage.,,pubmed:29700229,,Zebrafish Embryos  4hpf,GSM3067189,,tissue:Zebrafish Embryo Dissociated Cells|strain:TU|developmental stage:04hpf|treatment:untreated embryos|genotype:wild type,Zebrafish Embryos  4hpf,"inDrops FASTQ files were demultiplexed using a custom python pipeline as previously described see Zilionis et al.  Nature Protocols 2017 and https://github.com/indrops/.  Each sequencing spot is associated with a single biological read and 1 3 technical reads  depending on the specific inDrops library preparation chemistry used.  FASTQs DEW001 DEW003 used V1 chemistry in which read2 is the biological read and read1 is the metadata read.  DEW010 DEW057 used V2 chemistry in which read1 is the biological read and read2 is the metadata read.  DEW101 208 used V3 chemistry in which read1 is the biological read  read2 carries the first half of the cell barcode  read3 carries the library index  and read4 carries the second half of the cell barcode and the UMI. Sequencing reads were first sorted according to sample of origin; Individual samples were then formatted as a single demultiplexed FASTQ in which single cell and UMI barcodes for each read are listed in the header. These FASTQ files are deposited in NCBI SRA and can be used to resume the inDrops.py read processing pipeline at step 2 ""Identify abundant barcodes""  see https://github.com/indrops/. Each cDNA read was trimmed using Trimomatic version 0.32; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:16. Cell specific barcodes for each cDNA read were then matched against a set of pre determined barcodes. Up to two nucleotide mismatch errors were corrected; other reads were discarded. cDNA reads were then aligned to a zebrafish transcriptome using Bowtie version 1.1.1  parameters:  n 1  l 15  e 200  m 200  best  strata  a. The reference transcriptome was built from the zebrafish GRCz10 genome assembly Accesion: GCF 000002035.5.Mapped reads were then processed into counts of UMI filtered transcripts per gene. UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by manually inspecting a weighted histogram of UMI counts for each cell barcode  and thresholding only the top 95% of the largest and often the only mode of the distribution. UMI counts matrices originating from the same biological sample were combined into a single table.  UMI counts were adjusted by a total counts normalization. For TracerSeq embryos  inDrops FASTQ files generated from GFP targeted sequencing libraries were demultiplexed as described above.  These FASTQ files were then parsed to generate TracerVSCellBarcodes tables using custom Matlab scripts see: https://github.com/wagnerde/TracerSeq. Genome build: GRCz10 Supplementary files format and content: Raw UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 Normalized UMI filtered counts csv. Matrix rows are transcripts  columns are single cells.  Column headers are in the following format: LibraryName CellBarcodePart1 CellBarcodePart2 ClusterIDs txt.  An array of clusterID assignments for each cell column in the associated CSV tables ClusterNames csv.  A table of annotations for each ClusterID. Convert DEW to SRR csv.  A table for converting between DEW and NCBI library names. CellTracerCounts csv.  A table where each row is a unique TracerSeq transcript barcode; column1: inDrops cell barcode; column2: TracerSeq clone # assignment; column3: corrected TracerSeq barcode sequence; column4: UMI counts.",Zebrafish Embryo Dissociated Cells,,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,,strain:TU|developmental stage:04hpf|treatment:untreated embryos|genotype:wild type,GSM3067189,GSM3067189: Zebrafish Embryos  4hpf; Danio rerio; RNA Seq,GSM3067189,,1,Zebrafish embryos were incubated to the indicated times post fertilization and chorions were removed by incubating in 1mg/mL Pronase Sigma P5147 1G for 3 4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl  2.1 mM KCl 1.2 mM MgSO4  1.8 mM CaNO32  15 mM HEPES   pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described Manoli & Driever  Cold Spring Harbor Protocols 2012 with the following specifications. Wild type and CRISPR targeted samples were each prepared from 20 100 embryos. Embryo tissues were triturated to homogeneity in 1 5mL FACSmax cell dissociation solution Genlantis T200100 and incubated for 4 5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh Fisher 352340  and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS no Ca/Mg  Life Technologies 14190 144 containing 1% BSA Sigma A3311 100G  and subjected to 2 3 additional rounds of centrifugation and resuspension.  post washing  cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium Sigma D1556 250ML.  Cell density was quantified manually using INCYTO™ C Chip™ Disposable Hemacytometers Fisher 22 600 100  and adjusted to 100 000 cells per mL.  For single embryo dissociations  all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes Eppendorf 022431005 that had been pre coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single cell transcriptomes were then barcoded using the inDrops platform as previously described Zilionis et al.  Nature Protocols 2017. Following the within droplet reverse transcription step  emulsions were split into batches of approximately 1 000 2 000 cells  frozen at  80C  and subsequently processed as individual RNA seq libraries. Single cell RNA Seq libraries were prepared using a protocol customized for the inDrops platform see Zilionis et al.  Nature Protocols 2017,GEO Accession:GSM3067189,RNA-Seq,TRANSCRIPTOMIC,cDNA,SINGLE,ILLUMINA,NextSeq 500,,SRP136369,,loader:fastq load.py|options:  appendBCtoName   platform=Illumina,DEW051.fastq.gz,fastq,2180698840.0,65470578.0,GSM3067189 r2,0:33.31,A:552236143;C:443942730;G:453015797;T:731500517;N:3653,33,,,,552236143,443942730,453015797,731500517,3653,SRX3841318,SRS3087478,SRA672434,GEO,"Systems Biology, Harvard Medical School",1,0.84649,,0.12592,,0.78295,,0.64894,,34,,B,,usable mapping rate,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_droplet,indrops,,United States,2018-03-23,Blastula,Embryo,Embryo Imprecise,All anatomical structures