{"database": "metadata", "table": "run_metadata", "rows": [[38478, "SRR1872980", "SRX914872", "SRS869430", "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\u2019s 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", null, "pubmed:25867923", null, "zf3 dis S345", "GSM1629105", null, "tissue:50% Epiboly stage|group:experimental batch 3", "zf3 dis S345", "To map reads  we slightly modified the Zv9 reference transcriptome  since our reads were expected to originate from the 5\u2019 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", null, "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 \u03bcl 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 \u03bcl of DMEM/F12 was added to dilute the cell mixture  then 120 \u03bcl 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 \u03bcl of media that contained a single cell. This was pipetted into 3 \u03bcl 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\u2019 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\u20135 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\u00b0C or 28\u00b0C until they reached 50% epiboly about 6 hpf at 28\u00b0C. At 50% epiboly  single embryos were visually confirmed to be at the correct stage", "group:experimental batch 3", "GSM1629105", "GSM1629105: zf3 dis S345; Danio rerio; RNA Seq", "GSM1629105", null, "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 \u03bcl 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 \u03bcl of DMEM/F12 was added to dilute the cell mixture  then 120 \u03bcl 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 \u03bcl of media that contained a single cell. This was pipetted into 3 \u03bcl 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\u2019 transcript end  retains strand information  and is compatible with standard Illumina sequencing primers.", "GEO Accession:GSM1629105", "RNA-Seq", "TRANSCRIPTOMIC", "cDNA", "PAIRED", "ILLUMINA", "Illumina HiSeq 2500", null, "SRP055996", null, "loader:fastq load.py", "zf3_dis_S345.R1.fq zf3_dis_S345.R2.fq", "fastq fastq", 19894100.0, 397882.0, "GSM1629105 r1", "0:25 1:25", "A:4978487;C:4785641;G:4877454;T:5178846;N:73672", 25, 25, null, null, 4978487, 4785641, 4877454, 5178846, 73672, "SRX914872", "SRS869430", "SRA246089", "GEO", "Satija Lab, New York Genome Center", 2, 0.80074, 0.73644, 0.28821, 0.27089, 0.87452, 0.89213, 0.68194, 0.75059, 25, 25, "B", "B", "biological fallback assumption", "illumina", "hiseq_era", "unknown", "poly_a", "nextera", "sc", "single_cell_plate", "smartseq", null, "United States", "2015-03-09", "Multi-stage", "Embryo", "Embryo Imprecise", "All anatomical structures"]], "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": ["38478"], "units": {}, "query_ms": 9.499921987298876}