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Sample GSM1608257 Query DataSets for GSM1608257
Status Public on Apr 22, 2016
Title PECAM1plusPDGFRaminExp1
Sample type SRA
Source name Embryonic stem cells
Organism Mus musculus
Characteristics strain: 129X1/SvJ x 129S1/Sv
cell type: undifferentiated ESC culture
passages: 13-17
cell subpopulation: PECAM1plusPDGFRamin
Treatment protocol Single-cell suspensions of ESCs were obtained by dissociating with 0cell dissociation buffer (Invitrogen) at 37°C. Cells were incubated with the primary antibodies in PBS for 30 min on ice and sorted using a FACSariaIII
Growth protocol ESC were maintained feeder-free on gelatin (EmbryoMax® 0.1% Gelatin Solution, ES-006-B; Millipore) coated plates, in Knockout DMEM (10829–018; Gibco), 20% Knockout Serum Replacement (KSR, 10828-028; Gibco), 2 mM L-glutamine (25030-024; Gibco), 1X minimal essential medium nonessential amino acids (11140-035; Gibco), penicillin (100 U/ml)/streptomycin(100 μg/ml) (15140-122; Gibco), 100 μM β-Mercaptoethanol (31350; Gibco) and 1,000 U/ml recombinant mouse leukemia inhibitory factor (ESGRO, ESG1107; Chemicon International).
Extracted molecule total RNA
Extraction protocol RNA from 100000 sorted events for each subpopulation was extracted by using the Mirneasy RNA micro kit (QIAGEN). RNA was amplified by using the Ovation® RNA Amplification System V2 (NUGEN).
RNA libraries were prepared for sequencing using standard Illumina protocols
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Data processing Illumina Casava1.8 software used for basecalling.
Low quality ends of sequence reads were trimmed using FastX 0.0.13. Adapters were trimmed with cutadapt 1.2.1. Low quality sequence reads were filtered using FastX 0.0.13 and ShortRead1.20.0. Contaminants were removed using bowtie 2.1.0.
Reads were mapped to the reference genome of Mus musculus (Mus musculus GRCm38.73) using Tophat v2.0.8b with parameters: --library-type fr-firststrand --min-intron-length 50 --max-intron-length 500000 --no-coverage-search --no-mixed --read-realign-edit-dist 3.
The number of reads in the alignments that overlap with the gene features was counted by htseq-count 0.5.4p3 with parameters: -m union --stranded=no -a 0 -t exon -i geneid. Genes for which all samples have less than 1 counts-per-million (absent genes) were filtered.
RPKM (Reads per kilobase of gene sequence and per Million reads of library size) was calculated as following: Counts for each gene were divided by the total number of counts (in million) and by the gene length (in kbp).
Genome_build: Mus musculus GRCm38.73
Supplementary_files_format_and_content: tab-delimited text files of matrix tables of raw counts and RPKM values for all samples
Submission date Feb 12, 2015
Last update date May 15, 2019
Contact name Antonio Lo Nigro
Organization name Ismett
Street address Via tricomi 5
City Palermo
ZIP/Postal code 90134
Country Italy
Platform ID GPL13112
Series (1)
GSE65884 PDGFRα+ cells in ESC cultures represent the in vitro equivalent of the pre-implantation primitive endoderm precursors
BioSample SAMN03342077
SRA SRX876081

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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