run_metadata: 38481

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
38481	SRR1872977	SRX914869	SRS869432	SRP055996	PRJNA277737	Spatial reconstruction of single cell gene expression	GSE66688	Transcriptome Analysis	Spatial localization is a key determinant of cellular fate and behavior but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast single cell RNA seq allows for deep profiling of cellular gene expression but established methods separate cells from their native spatial context. Here we present Seurat a computational strategy to infer cellular localization by integrating single cell RNA seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish Danio rerio embryos inferring a transcriptome wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches and used it to identify a set of archetypal expression patterns and spatial markers. Additionally Seurat correctly localizes rare subpopulations accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. Overall design: We generated single cell RNA seq profiles from dissociated cells from developing zebrafish embryos late blastula stage 50% epiboly		pubmed:25867923		zf3 dis S1800	GSM1629102		tissue:50% Epiboly stage\|group:experimental batch 3	zf3 dis S1800	To map reads we slightly modified the Zv9 reference transcriptome since our reads were expected to originate from the 5’ end of each mRNA molecule which could cause problems if the reference gene models had an incorrect annotation for the transcription start site TSS. To address this we extended the TSS for all Zv9 genes by 100 bases upstream to allow for minor fluctuations. We then aligned read pairs directly to this modified reference transcriptome using Bowtie with the following parameters: q phred33 quals n 2 l 25 I 1 X 2000 a m 200. As expected following mapping our reads overwhelmingly originated from near the TSS. To quantify gene expression we leveraged the 5 bp Random Molecular Tags RMTs that were associated with each sequencing read pair. For each annotated gene in Zv9 we identified all read pairs that mapped to the correct strand and collected each of the 5 bp RMTs that were associated with these reads. We collapsed duplicate RMT sequences together in order to calculate the number of distinct RMTs associated with each gene. We quantified gene expression for 1 152 single cell libraries and identified a subset of 207 failed/low quality libraries with poor transcriptome complexity < 2 000 genes detected per cell. post excluding these remaining dataset of 945 cells on average we identified 47 000 unique molecules per sequencing library corresponding to the detection of 3 400 genes per single cell. Supplementary files format and content: zdata.expMatrix.txt Expression matrix for all samples	50% Epiboly stage		Two pairs of watchmaker forceps were used to dissect the blastula cap of the embryo away from the yolk. First one pair of forceps was used to hold and rotate the cap while the other was used to cut and pinch away the yolk that extended below the blastula cap. Then the blastula cap was cut slightly up the side which exposed the yolk that was inside of the blastula cap which could then be gently peeled away. The blastula cap was transferred by pipette into a microfuge tube that contained 60 μl of DMEM/F12 media. The cells were dissociated by vigorously flicking the tube 10 times and then pipetting the entire volume twice while visually confirming that dissociation had occurred. A timer was started at this time to track the amount of time the embryo had been dissociated. If cell clumps were still visible the tube was flicked again. 180 μl of DMEM/F12 was added to dilute the cell mixture then 120 μl of the diluted cell mixture was pipetted across the surface of a new agarose coated dish filled with DMEM/F12 media. To collect cells a P2 pipettor was used while observing the cells under the dissecting scope to collect 0.5 μl of media that contained a single cell. This was pipetted into 3 μl of TCL lysis buffer Qiagen in the lid of a PCR strip and mixed 3 times to ensure that lysis occurred. post collection of 8 cells the entire strip of lids was snapped into a 96 well plate and kept on dry ice. We used a modified strand specific single cell RNA seq protocol based on the SMART template switching method where the template switch oligonucleotide included a stretch of 5 randomized nucleotides thereby tagging each mRNA molecule with a random molecular tag RMT prior to PCR to mitigate amplification bias. Furthermore we used a modified library preparation protocol that shares similarities with Soumillon et al. and is based on the Nextera Sample Preparation Kit. It selectively amplifies the 5’ transcript end retains strand information and is compatible with standard Illumina sequencing primers.	Fertilized eggs from TL/AB in crosses were incubated in 1 mg/ml pronase Protease from Streptomyces griseus Sigma Aldrich in a glass dish for 4–5 minutes until the chorion began to blister. The embryos were submerged in 200 ml of embryo medium in a glass beaker without xxx them to contact air or plastic both of which will cause the embryos to burst. The medium was poured off and new medium was vigorously added again without xxx embryos to contact air twice in order to mechanically remove the chorions. The embryos were cultured in petri dishes coated with 2% agarose to avoid contact with plastic either at 23°C or 28°C until they reached 50% epiboly about 6 hpf at 28°C. At 50% epiboly single embryos were visually confirmed to be at the correct stage	group:experimental batch 3	GSM1629102	GSM1629102: zf3 dis S1800; Danio rerio; RNA Seq	GSM1629102		1	Two pairs of watchmaker forceps were used to dissect the blastula cap of the embryo away from the yolk. First one pair of forceps was used to hold and rotate the cap while the other was used to cut and pinch away the yolk that extended below the blastula cap. Then the blastula cap was cut slightly up the side which exposed the yolk that was inside of the blastula cap which could then be gently peeled away. The blastula cap was transferred by pipette into a microfuge tube that contained 60 μl of DMEM/F12 media. The cells were dissociated by vigorously flicking the tube 10 times and then pipetting the entire volume twice while visually confirming that dissociation had occurred. A timer was started at this time to track the amount of time the embryo had been dissociated. If cell clumps were still visible the tube was flicked again. 180 μl of DMEM/F12 was added to dilute the cell mixture then 120 μl of the diluted cell mixture was pipetted across the surface of a new agarose coated dish filled with DMEM/F12 media. To collect cells a P2 pipettor was used while observing the cells under the dissecting scope to collect 0.5 μl of media that contained a single cell. This was pipetted into 3 μl of TCL lysis buffer Qiagen in the lid of a PCR strip and mixed 3 times to ensure that lysis occurred. post collection of 8 cells the entire strip of lids was snapped into a 96 well plate and kept on dry ice. We used a modified strand specific single cell RNA seq protocol based on the SMART template switching method where the template switch oligonucleotide included a stretch of 5 randomized nucleotides thereby tagging each mRNA molecule with a random molecular tag RMT prior to PCR to mitigate amplification bias. Furthermore we used a modified library preparation protocol that shares similarities with Soumillon et al. and is based on the Nextera Sample Preparation Kit. It selectively amplifies the 5’ transcript end retains strand information and is compatible with standard Illumina sequencing primers.	GEO Accession:GSM1629102	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	Illumina HiSeq 2500		SRP055996		loader:fastq load.py	zf3_dis_S1800.R1.fq zf3_dis_S1800.R2.fq	fastq fastq	10790700.0	215814.0	GSM1629102 r1	0:25 1:25	A:2721576;C:2556263;G:2606994;T:2865815;N:40052	25	25			2721576	2556263	2606994	2865815	40052	SRX914869	SRS869432	SRA246089	GEO	Satija Lab, New York Genome Center	2	0.76882	0.8569	0.22815	0.24284	0.90851	0.88925	0.73921	0.67204	25	25	B	B	biological fallback assumption	illumina	hiseq_era	unknown	poly_a	nextera	sc	single_cell_plate	smartseq		United States	2015-03-09	Multi-stage	Embryo	Embryo Imprecise	All anatomical structures