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|Public on Jun 24, 2016
|sorted hematopoietic stem cells [JC4]
|cell type: bone marrow cells
cell subtype: hematopoietic stem cells
|Single hematopoietic stem cells were sorted (Lineage- Kit+ Sca1+ CD150+ CD48-) into Trizol (Life Technologies) and total RNA was isolated according to the manufacturer’s instructions, with the following alterations. RNA was precipitated overnight at -20 °C, with 2.5µg GlycoBlue (Life Technologies).
RNA was processed using the previously described CEL-seq technique, with the indicated modifications. Libraries were sequenced on an Illumina HiSeq2500 using 50bp paired end sequencing or Illumina NextSeq500 using 75bp paired end sequencing.
|Illumina HiSeq 2500
|Paired end reads were aligned to the transcriptome using bwa. Paired end reads obtained by CEL-seq were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all RefSeq gene models based on the mouse genome release mm10 downloaded from the UCSC genome browser and contained 31,109 isoforms derived from 23,480 gene loci. All isoforms of the same gene were merged to a single gene locus. The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first eight bases correspond to the cell specific barcode followed by 4 bases representing the unique molecular identifier. The remainder of the left read contains a polyT stretch followed by few (<15 transcript derived bases). The left read was not used for quantification. For each cell barcode we counted the number of unique molecular identifiers for every transcript and aggregated this number across all transcripts derived from the same gene locus. Based on binomial statistics we converted the number of observed unique molecular identifiers into transcript counts.
Supplementary_files_format_and_content: Tab separated data file for each sequencing library, listing all genes (rows) and the number of sequenced transcripts for all samples. Column name indicate the library, followed by micro-structure number, and cell number within the micro-structure. Column names containing "HSC" are single sorted hematopoietic stem cells. The rest are micro-dissected bone-marrow cells. The first column lists the official gene symbol followed by the chromosome name, separated by a double underscore.
|Mar 01, 2016
|Last update date
|May 15, 2019
|A. van Oudenaarden
|Single-cell RNA-seq of sorted and micro-dissected mouse bone marrow cells
|Transcriptomics of single-cell and bulk sorted and micro-dissected mouse bone marrow, fetal liver and small intestine crypt cells