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Sample GSM5111203 Query DataSets for GSM5111203
Status Public on Jun 23, 2021
Title Chromatin-associated total RNA Fp 3h
Sample type SRA
 
Source name mESC
Organism Mus musculus
Characteristics cell type: mESC E14
spike-in: Drosophila S2 RNA
treatment: 10 microM flavopiridol for 3 hr
Treatment protocol For Pol II inhibition experiments, cells were treated with triptolide (10 µM, Sigma T3652), flavopiridol (10 µM, Sigma F3055) for 1, 3 or 9 hrs. For RNA degradation experiments, cells were permeabilized with 0.05% Tween-20 in PBS for 10 min on ice, washed once, resuspended with PBS and mock-treated with 1 U/μl SUPERase.In (Invitrogen AM2694) or treated with 1 μg/μl RNaseA (Sigma R6513) for 30 min at RT.
Growth protocol mESC E14 cells were cultured on 0.1% gelatin-coated surfaces with KO-DMEM medium, 10% FCS, 5% knockout serum replacement, nonessential amino acids, L-glutamine, 2-mercaptoethanol, antibiotics (Invitrogen), and 1000 U/ml of leukaemia inhibitory factor (Abcam). Drosophila S2 cells (Thermofisher, R69007) were grown in Schneider's Drosophila Medium (ThermoFisher, Cat.no. 21720-02) supplemented with heat inactivated FBS (ThermoFisher, 10500064) and 25 U/mL of Penicillin-Streptomycin (ThermoFisher 15140-122).
Extracted molecule total RNA
Extraction protocol Trizol extraction protocol. For WCE RNA, 5x106 cells were resuspended in Trizol. For chromatin-associated RNAs, 15x106 cells were fractionated using the method published in Werner & Ruthenburg, 2015, and the chromatin fraction resuspended in Trizol.
For chromatin-associated total RNA sample, rRNA was depleted and libraries were prepared for sequencing of intronic and exonic RNA. For PolyA+ samples, libraries were prepared by selecting polyA+ RNAs.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description Libraries total depleted
All_counts.xlsx
Chr-fp-3h-total_S166
Data processing Fastq files were aigned to a combined Mouse-Drosophila genome assembly using STAR
Unique reads were extracted and the bam file was split into Mouse bam and Drosophila bam.
Number of reads mapping to Drosophila were used to calculate the scaling/normalization factor for the Mouse reads as shown in Orlando et al, 2014, which was then used to generate normalized bigwigs for each sample
featureCounts was used to get the counts of reads in gene for both Drosophila and Mouse genome
DESeq2 was used to calculate size factors for Drosophila reads which were then used to normalize the Mouse reads per sample
Genome_build: mm9 and BDGP5.25
Supplementary_files_format_and_content: Excel file with raw counts for exon/intron per gene for the three datasets (Chromatin-total, Chromatin-polyA and WCE-polyA)
 
Submission date Feb 26, 2021
Last update date Jun 23, 2021
Contact name Richard Jenner
E-mail(s) r.jenner@ucl.ac.uk
Organization name UCL Cancer Institute
Department Cancer Biology
Lab Regulatory Genomics
Street address 72 Huntley Street
City London
ZIP/Postal code WC1E 6BT
Country United Kingdom
 
Platform ID GPL24247
Series (1)
GSE150677 Nascent RNA antagonises the interaction of a set of regulatory proteins with chromatin
Relations
BioSample SAMN18063093
SRA SRX10175676

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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