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| Status |
Public on Jun 13, 2018 |
| Title |
RNA-seq E14 chromatin_2 |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 cell line
cell compartment: chromatin
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| 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).
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| 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)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sample 2
nascent RNA
processed data file: raw_counts_combined_chromatin_RNA-seq_samples.txt
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| 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
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| Submission date |
Mar 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Tessarz |
| E-mail(s) |
ptessarz@age.mpg.de
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| Organization name |
Max Planck Institute
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| Street address |
Joseph-Stelzman-Str 9b
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| City |
Cologne |
| State/province |
Nordrhein-Westfalen |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE90906 |
Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells |
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| Relations |
| BioSample |
SAMN06641997 |
| SRA |
SRX2671549 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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