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| Status |
Public on Mar 24, 2021 |
| Title |
PRO-seq with no estradiol treatment of MCF-7 cells and with shHP1a_shHP1b rep2 |
| Sample type |
SRA |
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| Source name |
PRO-seq with no estradiol treatment of MCF-7 cells and with shHP1a_shHP1b
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
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| Treatment protocol |
For estrogen stimulation, these cells were cultured in phenol-red free DMEM with 5% charcoal stripped FBS for at least 3 days and then treated with 100nM 17β-estradiol (E2) for 40 minutes. Control samples were treated with ethanol.
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| Growth protocol |
MCF-7 cells were cultured in DMEM, 10% FBS and 1% penicillin-streptomycin in a humidified incubator with 5% CO2 at 37C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Precision Run-on sequencing (PRO-seq) experiments were performed as previously described . Briefly, MCF-7 cells, for nuclei isolation, were incubated with swelling buffer (10 mM Tris-Cl pH7.5, 2 mM MgCl2, 3 mM CaCl2) for 5min on ice and then incubated with lysis buffer (swelling buffer with 0.5% NP-40 and 10% glycerol) for 5 min on ice, before being re-suspended in 100μl of freezing buffer (50 mM Tris-Cl pH8.0, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). For Run-on assay, an equal volume of reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 300 mM KCl, 1 mM DTT, 20 units of SuperaseIn, 1% sarkosyl, 500 μM ATP, GTP, bio-UTP and bio-CTP) was added into each sample before incubation at 30°C for 5 min. The nuclear run-on RNA was then extracted with TRIzol LS reagent (Invitogen) and subjected to hydrolysis, buffer exchange and purification by streptavidin beads (Thermo Fisher). Purified RNA was treated with PNK before being used for cDNA synthesis by using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® Kit (NEB). Obtained cDNA template was amplified by PCR using the Phusion High-Fidelity enzyme (NEB) for deep sequencing.
The library construction protocol followed the manufacturer’s instructions.
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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: PRO-seq
The image analysis and base calling were performed by using Illumina's Genome Analysis pipeline.
Quality control: FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and TRIMMOMATIC (http://www.usadellab.org/cms/?page=trimmomatic) was used to inspect the quality of the sequencing reads, and to trim the sequencing adaptors, if necessary.
Read alignment: Bowtie2 (Langmead et al., Nature Methods 2012) was employed to align PRO-seq samples to hg38 genome, and we have kept 1 copy/read per genome position.
Read counting: we have used the HOMER scripts analyzeRNA.pl and analyzeRepeats.pl in order to estimate the raw counts per gene in POL2-seq and PRO-seq data, and to compute the Pausing Ratio. The aligned reads were counted over the RefSeq gene bodies (after excluding a TSS 400bp-proximal region downstream of TSS).
Generation of bedgraph files: we have used the HOMER scripts makeUCSCfile and makeMultiWigHub.pl in order to generate bedgraph files for visualization in the UCSC genome browser.
Genome_build: hg38
Supplementary_files_format_and_content: bedgraph
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| Submission date |
Oct 21, 2019 |
| Last update date |
Mar 24, 2021 |
| Contact name |
MICHAEL G ROSENFELD |
| E-mail(s) |
[email protected]
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| Organization name |
HHMI/UCSD
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE139138 |
ChIRC-seq Reveals Promoter Antisense RNAs and HP1α as Key Regulators of Promoter Pause Release and of Enhancer : Architectural RNA Interactions [PRO-seq] |
| GSE139199 |
Shape of promoter antisense RNAs regulates ligand-induced transcription activation |
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| Relations |
| BioSample |
SAMN13067253 |
| SRA |
SRX7026909 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4131796_PROseq.R30_shHP1ab_rep2.RM030.RM-030_S2_L003_R1_001.fastq.gz.after.trimmomatic.fastq.gz.bed.srm.dir.ucsc.bedGraph.gz |
70.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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