{"database": "metadata", "table": "run_metadata", "rows": [[36252, "SRR34017910", "SRX29213468", "SRS25409913", "SRP592701", "PRJNA1278741", "Single cell and in situ spatial analyses reveal the diversity of newly born hematopoietic stem cells and of their niches [scRNA Seq]", "GSE300039", "Transcriptome Analysis", "Hematopoietic stem cells HSCs and more committed progenitors collectively referred to as HSPCs emerge from vessels during development  via Endothelial to Hematopoietic Transition EHT. Recently  using the zebrafish embryo  we showed that two EHT cell types emerge from the dorsal aorta  raising the question of their subsequent fate. To address this issue  we established a complex pipeline based on single cell photoconversion and transgenic lines to characterize the abilities of EHT cell progenies to conquer hematopoietic organs and to obtain their transcriptomic profiles. We show that the two EHT cell types lead to partly differentially fated cells  with significant differences in thymus colonization and T lymphoid lineage commitment. In addition  we investigated implantation of HSPCs in niches  with the support of HSPC signatures gata2b and cd34/podocalyxin  retrieved from our single cell datasets. This revealed  at unprecedented resolution  the homing of HSPCs in niches of entire early larvae  including the pronephros  the sub aortic and caudal regions  as well as the area contacting the supra intestinal artery. Our work provides new insights on fundamental aspects of HSPC fate acquisition  from their emergence to their homing in specific niches. Overall design: Single cell transcriptomics analysis of newly born hematopoietic stem and progenitor cells. This dataset comprises progenies of cells photoconverted at 2dpf and sorted by FACS at 5dpf  using the Tgkdrl:nls kikume transgenic line  as well as sorted FACS hematopoietic cells from transgenic reporter lines  namely from the Tgrunx1+23:eGFP and the outcross of the Tgcd41:eGFP and Tggata2b:RFP lines. Larvae were collected post dissection separating the rostral  anterior and posterior region along a transversal section at the posterior limit of the elongated yolk and at the posterior limit of the swim bladder  dissociation and sorting using a BD FACS AriaIII cell sorter. Progenies of photoconverted cells comprise progenies of single EHT photoconversion either EHT pol+ or EHT pol  or whole hemogenic endothelium photoconversion. Cells were sorted in 384 wells plates 28 plates  in 2 independent sessions of 14 plates each and the transcriptomic library was prepared according to the MARS seq protocol 2.0 Keren Shaul et al. 2019. Libraries were then sequenced using the NextSeq 500/550 High Output Kit v2 75 cycles Illumina. Raw reads were converted to fastq files using bcl2fastq package 2.20.0. Reads were demultiplexed and mapped to the Lawson Lab curated genome [Lawson et al.  2020  v4.3.2] using the MARS seq pipeline scripts Keren Shaul et al.  2019.", null, "pubmed:40501392", null, "5 dpf hematopoietic zebrafish cells  session 2", "GSM9052756", null, "tissue:sorted hematopoietic cells|cell type:sorted hematopoietic cells|geo loc name:missing|collection date:missing", "5 dpf hematopoietic zebrafish cells  session 2", "Raw reads were converted to fastq files using bcl2fastq package 2.20.0. Reads were demultiplexed and mapped to the Lawson Lab curated genome [Lawson et al.  2020  v4.3.2] using the MARS seq pipeline scripts Keren Shaul et al.  2019 Assembly: zebrafish genome Lawson lab version 4.3.2. Supplementary files format and content: Tab separated value files", "sorted hematopoietic cells", null, "Single cell suspension for FACS sorting was prepared using an optimized protocol adapted from multiple sources Bresciani et al.  2018; Manoli and Driever  2012; Samsa et al.  2016. All steps were performed with cooled solutions 4\u00b0C  and samples were kept on ice throughout the processing to preserve the viability of cells. Briefly  5dpf larvae were anesthetized using balneation in embryo medium supplemented with tricaine  at a final concentration of 640 \u00b5g/ml. Larvae between 20 and 30 whole larvae or 60 80 dissected segments per tube were washed twice in PBS before centrifugation 300 x g for 1 min @4\u00b0C and the supernatant was discarded. Larvae were incubated in 1 ml TrypLE medium Life Technologies  Cat#: 12605 010 for 10 min on ice  with gentle pipetting every 3 mins  first with a P1000 pipette and subsequently with a P200 pipette. Samples were then centrifugated for 7 min at 300g  before the TrypLE was removed and the cells were resuspended in 500 \u00b5l FACSmax medium Genlantis  Cat#: T200100. Ultimately  samples were passed through a 40 \u03bcm cell strainer moistened with FACSmax buffer onto a 35 mm cell culture dish using a syringe plunger. The cell strainer and the dish were washed with 300 \u00b5l of FACS max and the flow through was transferred to a FACS tube and kept on ice until sorting within the hour. Cell sorting was performed on a BD FACS AriaIII cell sorter. For all samples  gating was done on SSC A vs. FSC A to collect cells  then on FSC W vs. FSC A to keep only singlets and on APC A to remove dead cells marked by Draq7 Thermofisher  Cat#: D15106. Cells were sorted in 384 wells plates 28 plates  in 2 independent sessions of 14 plates each and the transcriptomic library was prepared according to the MARS seq protocol 2.0 Keren Shaul et al. 2019. Libraries were then sequenced using the NextSeq 500/550 High Output Kit v2 75 cycles Illumina. Raw reads were converted to fastq files using bcl2fastq package 2.20.0. Reads were demultiplexed and mapped to the Lawson Lab curated genome [Lawson et al.  2020  v4.3.2] using the MARS seq pipeline scripts Keren Shaul et al.  2019. Briefly  a first step of reverse transcription RT SuperScript III  Thermo Fisher  Cat#: 18080085 was conducted to barcode the transcripts with unique cell and UMI barcodes. post RT  primers were removed with an exonuclease ExoI NEB  Cat#: M0293L  and the barcoded cDNAs for each single cell in a half plate were pooled together for the next steps. cDNA was converted to dsDNA Second Strand synthesis kit  NEB  Cat#: E611L and linearly amplified by in vitro transcription HiScribe T7 High Yield RNA Synthesis Kit  NEB  Cat#: E2040S. The dsDNA was degraded TURBO DNase Fisher  Cat#: 10646175 and the amplified RNA fragmented for sequencing Fisher  Cat#: 10426914. A pool barcode with Illumina adapters for each half plate was added by RNA DNA ligation before a second RT step AffinityScript Multiple Temperature reverse transcriptase  Agilent  Cat#: 600109. Final amplification by PCR was performed KAPA HiFi HotStart ReadyMix  Roche Diagnostics  Cat#: 7958935001. The resulting libraries were controlled by qPCR amplification of housekeeping genes ef1a and \u00df actin  DNA quality control using a TapeStation  Agilent and DNA concentration using the Qubit dsDNA high sensitivity assay.", null, "cell type:sorted hematopoietic cells", "GSM9052756", "GSM9052756: 5 dpf hematopoietic zebrafish cells  session 2; Danio rerio; RNA Seq", "GSM9052756 r1", "GSM9052756", "1", "Single cell suspension for FACS sorting was prepared using an optimized protocol adapted from multiple sources Bresciani et al.  2018; Manoli and Driever  2012; Samsa et al.  2016. All steps were performed with cooled solutions 4\u00b0C  and samples were kept on ice throughout the processing to preserve the viability of cells. Briefly  5dpf larvae were anesthetized using balneation in embryo medium supplemented with tricaine  at a final concentration of 640 \u00b5g/ml. Larvae between 20 and 30 whole larvae or 60 80 dissected segments per tube were washed twice in PBS before centrifugation 300 x g for 1 min @4\u00b0C and the supernatant was discarded. Larvae were incubated in 1 ml TrypLE medium Life Technologies  Cat#: 12605 010 for 10 min on ice  with gentle pipetting every 3 mins  first with a P1000 pipette and subsequently with a P200 pipette. Samples were then centrifugated for 7 min at 300g  before the TrypLE was removed and the cells were resuspended in 500 \u00b5l FACSmax medium Genlantis  Cat#: T200100. Ultimately  samples were passed through a 40 \u03bcm cell strainer moistened with FACSmax buffer onto a 35 mm cell culture dish using a syringe plunger. The cell strainer and the dish were washed with 300 \u00b5l of FACS max and the flow through was transferred to a FACS tube and kept on ice until sorting within the hour. Cell sorting was performed on a BD FACS AriaIII cell sorter. For all samples  gating was done on SSC A vs. FSC A to collect cells  then on FSC W vs. FSC A to keep only singlets and on APC A to remove dead cells marked by Draq7 Thermofisher  Cat#: D15106. Cells were sorted in 384 wells plates 28 plates  in 2 independent sessions of 14 plates each and the transcriptomic library was prepared according to the MARS seq protocol 2.0 Keren Shaul et al. 2019. Libraries were then sequenced using the NextSeq 500/550 High Output Kit v2 75 cycles Illumina. Raw reads were converted to fastq files using bcl2fastq package 2.20.0. Reads were demultiplexed and mapped to the Lawson Lab curated genome [Lawson et al.  2020  v4.3.2] using the MARS seq pipeline scripts Keren Shaul et al.  2019. Briefly  a first step of reverse transcription RT SuperScript III  Thermo Fisher  Cat#: 18080085 was conducted to barcode the transcripts with unique cell and UMI barcodes. post RT  primers were removed with an exonuclease ExoI NEB  Cat#: M0293L  and the barcoded cDNAs for each single cell in a half plate were pooled together for the next steps. cDNA was converted to dsDNA Second Strand synthesis kit  NEB  Cat#: E611L and linearly amplified by in vitro transcription HiScribe T7 High Yield RNA Synthesis Kit  NEB  Cat#: E2040S. The dsDNA was degraded TURBO DNase Fisher  Cat#: 10646175 and the amplified RNA fragmented for sequencing Fisher  Cat#: 10426914. A pool barcode with Illumina adapters for each half plate was added by RNA DNA ligation before a second RT step AffinityScript Multiple Temperature reverse transcriptase  Agilent  Cat#: 600109. Final amplification by PCR was performed KAPA HiFi HotStart ReadyMix  Roche Diagnostics  Cat#: 7958935001. The resulting libraries were controlled by qPCR amplification of housekeeping genes ef1a and \u00df actin  DNA quality control using a TapeStation  Agilent and DNA concentration using the Qubit dsDNA high sensitivity assay.", null, "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "cDNA", "PAIRED", "ILLUMINA", "NextSeq 500", null, "SRP592701", null, null, "TORCQ_2023_S0_L004_R1_001.fastq.gz TORCQ_2023_S0_L004_R2_001.fastq.gz", "fastq fastq", 9409346870.0, 122199310.0, "GSM9052756 r4", "0:57 1:20", "A:2048111796;C:1451606983;G:1595602008;T:1870010070;N:2444016013", 57, 20, null, null, 2048111796, 1451606983, 1595602008, 1870010070, 2444016013, "SRX29213468", "SRS25409913", "SRA2151098", "Institut Pasteur", "Institut Pasteur", null, null, null, null, null, null, null, null, null, null, null, null, null, null, "illumina", "nextseq", "unknown", "random_priming", "unknown", "sc", "single_cell_plate", "marsseq", null, "France", "2025-06-17", "Larval", "Larval", "Blood", "Hematopoietic System"]], "columns": ["rowid", "run.accession", "experiment.accession", "sample.accession", "study.accession", "bioproject", "study.title", "study.alias", "study.type", "study.abstract", "study.attributes", "study.PMIDs", "sample.description", "sample.title", "sample.alias", "sample.centername", "sample.attributes", "GEOsample.title", "GEOsample.dataprocessing", "GEOsample.source", "GEOsample.treatmentprotocol", "GEOsample.extractprotocol", "GEOsample.growthprotocol", "GEOsample.characteristics", "GEOsample.accession", "experiment.title", "experiment.alias", "experiment.library_name", "experiment.design_description", "experiment.library_construction_protocol", "experiment.attributes", "experiment.library_strategy", "experiment.library_source", "experiment.library_selection", "experiment.library_layout", "experiment.platform", "experiment.instrument_model", "experiment.spot_descriptor", "experiment.study_ref", "run.title", "run.attributes", "run.filename", "run.semantic_name", "run.total_bases", "run.total_spots", "run.alias", "run.read_lengths", "run.base_counts", "run.r1_length", "run.r2_length", "run.r3_length", "run.r4_length", "run.Acount", "run.Ccount", "run.Gcount", "run.Tcount", "run.Ncount", "run.experiment", "run.pool_member", "submission.accession", "submission.srasource", "submission.bioprojectsource", "seqdetective.n_mates", "seqdetective.mapping_rate.mate1", "seqdetective.mapping_rate.mate2", "seqdetective.nofeature_rate.mate1", "seqdetective.nofeature_rate.mate2", "seqdetective.sparsity.mate1", "seqdetective.sparsity.mate2", "seqdetective.pos_strand_rate.mate1", "seqdetective.pos_strand_rate.mate2", "seqdetective.readlen.mate1", "seqdetective.readlen.mate2", "seqdetective.judgement.mate1", "seqdetective.judgement.mate2", "seqdetective.judgement.reason", "platform_family", "instrument_generation", "read_bias", "selection_class", "prep_kit", "sc_or_bulk", "tech_class", "technology", "tech_variant", "submission.bioprojectsource.country", "earliest_date", "devstage_curation", "devstage_curation_coarse", "tissue_curation", "tissue_curation_coarse"], "primary_keys": ["rowid"], "primary_key_values": ["36252"], "units": {}, "query_ms": 11.675891000777483}