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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jun 23, 2021 |
Title |
Chromatin-associated total RNA Fp 1h |
Sample type |
SRA |
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Source name |
mESC
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Organism |
Mus musculus |
Characteristics |
cell type: mESC E14 spike-in: Drosophila S2 RNA treatment: 10 microM flavopiridol for 1 hr
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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.
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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).
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
Libraries total depleted All_counts.xlsx Chr-fp-1h-total_S165
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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)
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Submission date |
Feb 26, 2021 |
Last update date |
Jun 23, 2021 |
Contact name |
Richard Jenner |
E-mail(s) |
r.jenner@ucl.ac.uk
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Organization name |
UCL Cancer Institute
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Department |
Cancer Biology
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Lab |
Regulatory Genomics
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Street address |
72 Huntley Street
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City |
London |
ZIP/Postal code |
WC1E 6BT |
Country |
United Kingdom |
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Platform ID |
GPL24247 |
Series (1) |
GSE150677 |
Nascent RNA antagonises the interaction of a set of regulatory proteins with chromatin |
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Relations |
BioSample |
SAMN18063094 |
SRA |
SRX10175675 |
Supplementary data files not provided |
SRA Run Selector |
Processed data are available on Series record |
Raw data are available in SRA |
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