run_metadata: 36252

This data as json

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
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.		pubmed:40501392		5 dpf hematopoietic zebrafish cells session 2	GSM9052756		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		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°C 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 µg/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°C 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 µl FACSmax medium Genlantis Cat#: T200100. Ultimately samples were passed through a 40 μm 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 µl 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 ß actin DNA quality control using a TapeStation Agilent and DNA concentration using the Qubit dsDNA high sensitivity assay.		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°C 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 µg/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°C 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 µl FACSmax medium Genlantis Cat#: T200100. Ultimately samples were passed through a 40 μm 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 µl 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 ß actin DNA quality control using a TapeStation Agilent and DNA concentration using the Qubit dsDNA high sensitivity assay.		RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	NextSeq 500		SRP592701			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			2048111796	1451606983	1595602008	1870010070	2444016013	SRX29213468	SRS25409913	SRA2151098	Institut Pasteur	Institut Pasteur															illumina	nextseq	unknown	random_priming	unknown	sc	single_cell_plate	marsseq		France	2025-06-17	Larval	Larval	Blood	Hematopoietic System