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Sample GSM2549739 Query DataSets for GSM2549739
Status Public on Jun 13, 2018
Title RNA-seq E14 chromatin_1
Sample type SRA
 
Source name mESCs
Organism Mus musculus
Characteristics cell line: E14 cell line
cell compartment: chromatin
Growth protocol The E14 cell line (mESCs) was cultured at 37 °C, 7.5% CO2, on 0.1% gelatin coated plates, in DMEM + GlutaMax™ (Gibco) with 15% fetal bovine serum (Gibco), MEM non- essential amino acids (Gibco), penicillin/streptomycin (Gibco), 550 µM 2-mercaptoethanol (Gibco), and 10 ng/ml of leukaemia inhibitory factor (LIF) (eBioscience).
Extracted molecule total RNA
Extraction protocol Cell fractionation was performed as described in (Pandya-Jones & Black, RNA, 2009), (Bhatt et al., Cell, 2012) and based on (Wuarin & Schibler, MCB, 1994) with modifications. All subsequent steps have been conducted on ice or at 4°C and in the presence of 25 µM α-amanitin (Sigma, A2263), 50 Units SUPERaseIN (Life Technologies, AM2696) and Protease inhibitors cOmplete (Roche, 11873580001) according to manufacturer’s instructions using RNAse free equipment. All buffers have been pre-chilled on ice before use. The cell pellet corresponding to 1x107 cells was gently resuspended in 200 µl cytoplasmic lysis buffer (0.15% NP-40, 10 mM Tris-HCl pH 7.0, 150 mM NaCl). The cell lysate was incubated for 5 min on ice, layered onto 500 µl sucrose buffer (10 mM Tris-HCl pH 7.0, 150 mM NaCl, 25% sucrose) and centrifuged at 16,000 g for 10 min. The supernatant corresponding to the cytoplasmic fraction was carefully removed.
Sequencing library preparation was performed as described in (Mayer & Churchman, 2016)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description Sample 1
nascent RNA
processed data file: raw_counts_combined_chromatin_RNA-seq_samples.txt
Data processing RNA-seq files were aligned using STAR (v 2.4.1b). Raw counts were obtained from featureCounts of the Rsubread package (R/Bioconductor).
Genome_build: mm10
Supplementary_files_format_and_content: text file with raw counts
 
Submission date Mar 24, 2017
Last update date May 15, 2019
Contact name Peter Tessarz
E-mail(s) ptessarz@age.mpg.de
Organization name Max Planck Institute
Street address Joseph-Stelzman-Str 9b
City Cologne
State/province Nordrhein-Westfalen
ZIP/Postal code 50931
Country Germany
 
Platform ID GPL17021
Series (1)
GSE90906 Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells
Relations
BioSample SAMN06641996
SRA SRX2671548

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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