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| Status |
Public on Jan 18, 2023 |
| Title |
mNET_seq_DMSO4h_Trip_0min_rep1 |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESCs
strain: 129/Ola
auxin treatment: DMSO
triptolide treatment: 0 min
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| Treatment protocol |
For promoter-proximal Pol II half-life experiments by mNET-seq, the pre-warmed fresh medium with 1 µM Triptolide (Sigma-Aldrich, T3652) was added to the dish to block transcription initiation. The sample was collected before the addition of QIAzol (Qiagen), which was used to stop the reaction at the desired time point.
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| Growth protocol |
E14 ESCs (129/Ola background) were maintained on 0.2% gelatin-coated plates in Glasgow Minimum Essential Medium (GMEM, Sigma-Aldrich, G5154) containing 15% fetal bovine serum (Gibco, 26140079), supplemented with 1× Pen-Strep (Thermofisher Scientific, 15140122), 2 mM Glutamax (Thermofisher Scientific, 35050061), 50 µM β-mercaptoethanol (Thermofisher Scientific, 21985023), 0.1 mM nonessential amino acids (Thermofisher Scientific, 11140050), 1 mM sodium pyruvate (Thermofisher Scientific, 11360070), and Leukemia Inhibitory Factor (LIF, 1000U/mL, Millipore).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were seeded the day before the experiment to get 100 million cells per sample the following day. We randomly assigned flasks for each treatment and treated cells with DMSO only or Auxin ligand, before extracting the chromatin-bound RNA Pol II. The cells were first washed with ice-cold DPBS, resuspended in 4 mL ice-cold HLB+N buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% (vol/vol) NP-40 and 1× proteinase inhibitor) and incubated on ice for 5 minutes, then cells were scrapped to a 15-mL centrifugate tube.
Libraries were constructed using NEBNext Multiplex Small RNA Library Prep kit (NEB, NC0477293).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
Library strategy: mNET-seq
Illumina bcl2fastq2 Conversion Software v2.20 used for basecalling.
Reads were demultiplexed, trimmed for adapter content with cutadapt and mapped with STAR to the GRCm38 (mm10) genome assembly.
Further data processing was carried out using the R/Bioconductor environment.
Genome_build: mm10
Supplementary_files_format_and_content: All processed data is made available as supplementary files to the manuscript
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| Submission date |
Aug 09, 2021 |
| Last update date |
Jan 18, 2023 |
| Contact name |
Hua Wang |
| E-mail(s) |
moodswh@gmail.com
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| Organization name |
Memorial Sloan Kettering Cancer Center
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| Street address |
430 East 67th Street
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (2) |
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| Relations |
| BioSample |
SAMN20669647 |
| SRA |
SRX11676027 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5509245_mNET_seq_DMSO4h_Trip_0min_rep1.bw |
88.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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