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Sample GSM5509245 Query DataSets for GSM5509245
Status Public on Jan 18, 2023
Title mNET_seq_DMSO4h_Trip_0min_rep1
Sample type SRA
 
Source name mESCs
Organism Mus musculus
Characteristics cell type: mESCs
strain: 129/Ola
auxin treatment: DMSO
triptolide treatment: 0 min
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.
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).
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).
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model NextSeq 550
 
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
 
Submission date Aug 09, 2021
Last update date Jan 18, 2023
Contact name Hua Wang
E-mail(s) moodswh@gmail.com
Organization name Memorial Sloan Kettering Cancer Center
Street address 430 East 67th Street
City New York
State/province New York
ZIP/Postal code 10065
Country USA
 
Platform ID GPL21626
Series (2)
GSE181686 mNET-seq on COMPASS-degron cells
GSE181714 COMPASS-degron cells
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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