run_metadata: 29473

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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
29473	SRR27352090	SRX23028809	SRS19990692	SRP480147	PRJNA1057153	Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion	GSE252032	Transcriptome Analysis	During differentiation cells become structurally and functionally specialized but comprehensive views of the underlying remodeling processes are elusive. Here we leverage scRNA seq developmental trajectories to reconstruct differentiation using two secretory tissues as a model system – the zebrafish notochord and hatch gland. First we present an approach MIMIR to integrate expression and functional similarities for gene module identification revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second we focused on the unfolded protein response UPR transducer module to study how general versus cell type specific secretory functions are regulated. By profiling loss and gain of function embryos we found that the UPR transcription factors creb3l1 creb3l2 and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program while xbp1 partners with bhlha15 to activate a gland specific secretion program. Our study offers an integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions. Overall design: scRNA seq experiments were conducted to study the regulatory roles of the unfolded protein response UPR pathway transcription factors TFs during zebrafish embryogenesis. We focused on the four UPR TFs xbp1 atf6 creb3l1 and creb3l2 that are expressed during the early differentiation of the notochord and/or the hatch gland. We hypothesized that the UPR TFs regulate the secretory machinery genes in these two cell types. To identify the endogenous target genes of the UPR TFs we performed scRNA seq of UPR TF loss of function embryos at 12 hpf when UPR TFs and secretory pathway genes are normally highly expressed in the notochord and hatch gland. Loss of function embryos were generated by CRISPR mediated mutagenesis. Crispants were created by injecting Cas9 protein and guide RNAs gRNAs into 1 cell stage embryos. We additionally raised a stable mutant line for creb3l1. Co injecting Cas9 with gRNA targeting multiple genes in wild type or the stable mutant embryos allowed testing for redundant functions by generating double triple and quadruple loss of function embryos. Tyrosinase tyr crispants which do not exhibit developmental defects were generated as the control. Using this loss of function dataset we defined target genes for UPR TFs as the genes whose expression levels changed in corresponding crispants and/or mutants. Next we performed scRNA seq on UPR TF gain of function embryos to test the sufficiency of the UPR TFs in regulating their endogenous targets. To generate gain of function embryos we globally mis expressed each UPR TF by injecting mRNAs encoding their activated forms into 1 cell stage embryos. We then performed scRNA seq on the resulting embryos at 8hpf when 1 a sizable population of transcriptionally distinct notochord and hatch gland cells are detectable and 2 the endogenous UPR TFs and secretory pathway genes are not yet highly expressed except for xbp1. Since xbp1 can be activated by UPR TFs including creb3l1 creb3l2 and atf6 and atf6 can be activated by xbp1 we mis expressed the UPR TFs in xbp1 or xbp1+atf6 double crispants to reduce potential indirect effects. Single cell transcriptomes were obtained for mCherry control the activated forms of xbp1 xbp1 s creb3l1 creb3l1 N creb3l2 creb3l2 N and atf6 atf6 N as well as the non activated full length creb3l2 creb3l2 full. All scRNA seq was performed using the 10x genomics platform V3 and V3.1.		pubmed:38234833;pubmed:39591963		atf6 N me 40pg	GSM7992594		source name:Whole embryo\|tissue:Whole embryo\|strain:TL/AB\|developmental stage:8 hpf\|genotype:atf6 N mis expression in xbp1 crispant\|geo loc name:missing\|collection date:missing	atf6 N me 40pg	The cellranger command line tool was used to map reads in the fastq files to reference transcriptomes with the "cellranger count" command For libraries that include xbp1 s misexpression ME E 5 to ME E 8 the reference transcriptome was modified: the mis expressed codon optimized xbp1 s sequence was appended to the reference transcriptome. Assembly: dr99 Supplementary files format and content: All processed data are output from the "cellranger count" command. barcodes.tsv.gz features.tsv.gz and matrix.mtx.gz can be collectively input into third party packages to allow users to wrangle the barcode feature matrix. The matrix contains only detected cell associated barcodes. Each element of the matrix is the number of UMIs associated with a feature row and a barcode column. The filtered feature bc matrix.h5 files contain the same detected cell associated feature and barcode information in HDF5 format.	Whole embryo	1 cell stage embryos were either injected with Cas9 and gRNA injected with mRNA or uninjected according to experimental conditions.	At desired developmental stages 8hpf or 12hpf approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl 1.8 mM KCl 1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5 110 mM NaCl 3.5 mM KCl 2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco 11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry several modifications were made to the dissociation protocol described above. Briefly post being transferred to 2ml Eppendorf tubes embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma P5380] 125 U/ml DNaseI 2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS 0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer’s solution. Centrifuging resuspending and filtering steps were then performed as in the previous protocol but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1	Zebrafish embryos were collected at 1 cell stage and cultured in standard E3 medium in a 27°C incubator until desired stages.	tissue:Whole embryo\|strain:TL/AB\|developmental stage:8 hpf\|genotype:atf6 N mis expression in xbp1 crispant	GSM7992594	GSM7992594: atf6 N me 40pg; Danio rerio; RNA Seq	GSM7992594 r1	GSM7992594	1	At desired developmental stages 8hpf or 12hpf approximately 30 embryos per sample were de chonrionated with pronase. For libraries prepared with 10x chromium Single Cell three prime V3 chemistry embryos for each condition were transferred into a 2ml eppendorf tube containing ice cold deyolking buffer buffer 55 mM NaCl 1.8 mM KCl 1.25 mM NaHCO3. Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer 10 mM Tris pH 8.5 110 mM NaCl 3.5 mM KCl 2.7 mM CaCl2 was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM gibco 11594426 with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell three prime V3.1 chemistry several modifications were made to the dissociation protocol described above. Briefly post being transferred to 2ml Eppendorf tubes embryos were first incubated in a protease solution 10 mg/ml BI protease [Sigma P5380] 125 U/ml DNaseI 2.5 mM EDTA in DPBS on ice for 4min. Embryos were then washed with a stop solution 30% FBS 0.875 mM CaCl2 in DPBS and gently triturated by pipetting in Ringer's solution. Centrifuging resuspending and filtering steps were then performed as in the previous protocol but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell three prime Reagent Kits V3 and V3.1		RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	PAIRED	ILLUMINA	Illumina NovaSeq 6000		SRP480147		loader:fastq load.py	ME_D_4_S7_L001_R2_001.fastq.gz ME_D_4_S7_L001_R1_001.fastq.gz ME_D_4_S7_L001_I1_001.fastq.gz	fastq fastq fastq	34201406232.0	271439732.0	GSM7992594 r1	0:8 1:28 2:90	A:6970137729;C:5129294511;G:5749174804;T:6578660562;N:2308274	8	28	90		6970137729	5129294511	5749174804	6578660562	2308274	SRX23028809	SRS19990692	SRA1775820	Schier Lab, Harvard University	Schier Lab, Harvard University	1	0.91553		0.16107		0.79835		0.5523		90		B		usable mapping rate	illumina	novaseq_era	full_length	cdna_unspecified	unknown	sc	single_cell_droplet	10x		United States	2023-12-26	Gastrula	Embryo	Whole Organism	All anatomical structures