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| Status |
Public on Jun 13, 2018 |
| Title |
NET-seq_Control_rep1 |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 cell line
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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 |
NET-seq libraries were prepared as previously described (Mayer & Churchman, 2016) with a few modifications. Briefly, chromatin associated nascent RNA was extracted through cell fractionation in the presence of α-amanitin, protease and RNAase inhibitors. > 90% recovery of ligated RNA and cDNA was achieved from 15 % TBE-Urea (Invitrogen) and 10% TBE-Urea (Invitrogen), respectively, by adding RNA recovery buffer (Zymo Research, R1070-1-10) to the excised gel slices and further incubating at 70°C (1500 rpm) for 15 min. Gel slurry was transferred through a Zymo-Spin IV Column (Zymo Research, C1007-50) and further precipitated for subsequent library preparation steps. cDNA containing the 3’ end sequences of a subset of mature and heavily sequenced snRNAs, snoRNAs, and rRNAs, were specifically depleted using biotinylated DNA oligos (Supplementary Table 6). Oligo-depleted circularised cDNA was amplified via PCR (5 cycles) and double stranded DNA was run on a 4% low melt agarose gel. The final NET-seq library running at ~150 bp was extracted and further purified using the ZymoClean Gel DNA recovery kit (Zymo Research)
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
nascent RNA
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| Data processing |
Library strategy: NET-seq
NET-seq files were aligned using STAR (v 2.4.2a). BAM files were converted to bigwig (1 bp bin), filtered by strand and normalised to x1 sequencing depth.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig
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| Submission date |
Mar 16, 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 |
SAMN06607727 |
| SRA |
SRX2646998 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2538816_NET.Control.rep1.forward.bw |
9.2 Mb |
(ftp)(http) |
BW |
| GSM2538816_NET.Control.rep1.reverse.bw |
8.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
| Processed data are available on Series record |
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