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| Status |
Public on Apr 25, 2022 |
| Title |
NRVM_PE_WCE |
| Sample type |
SRA |
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| Source name |
NRVM
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague Dawley
tissue: heart ventricles
age: post natal day 2-3
chip antibody: None
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| Treatment protocol |
48 hours of treatment: 1. Vehicle ; 2. PE (100 μM); 3. PE (100 μM)+THZ1(10 nM)
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| Growth protocol |
Neonatal rat ventricular myocyte (NRVM) were isolated from the hearts of 2-3 day old Sprague-Dawley rat pups from Charles River and maintained under standard conditions as described previously3. After overnight digestion with 0.25% trypsin at 4°C, cells were dissociated from tissue through series of digestion with 300 U/mL collagenase II (Worthington Biochemical Corporation, Lakewood, NJ). The dissociated cells were then preplated for 1.5 hours in cell culture dishes followed by 24 hours exposure to BrdU in culture medium to remove contaminating nonmyocytes and inhibit cardiomyocyte proliferation. Unless otherwise stated, NRVM were plated at a density of 105 cells/mL. Cells were initially plated in growth medium (DMEM supplemented with 5% FBS, 100 U/ml penicillin-streptomycin and 2 mM L-glutamine) for 48 hours. Prior to stimulation with agonists, NRVM were maintained in serum-free medium ((DMEM supplemented with 0.1% BSA, 1% insulin-transferrin-selenium supplement (Sigma I3146), 100 U/ml penicillin-streptomycin, and 2 mM L-glutamine) for additional 48 hours.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
NRVM were plated in 15 cm dishes at 4 x 10^6 cells/dish. Total of 15 million cells were used for each immunoprecipitation. After indicated treatments, cells were crosslinked with 1% formaldehyde for 10 min at room temperature followed by quenching with 0.125M glycine for 5 min. Cells were then lysed and chromatin was extracted. Isolated chromatin was subjected to shearing with Bioruptor (Diagenode) for 16 cycles (30sec on and 30 sec off/cycle) at high intensity. Small volume of sheared chromatin (50 uL) from each was stored at -80 °C as input. The rest of sheared chromatin was then incubated with 5mg RNAPII antibody (clone 8WG16, BioLegend 920102 lot B217159) with 50uL Dynabeads (Invitrogen) overnight at 4 °C. Chromatin was washed, eluted and reverse crosslinked overnight at 65 °C. Genomic DNA was purified from both immunoprecipitated samples and input samples.
The library was prepared with TrueSeq ChIP kit (Illumina)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Each ChIP-seq sample was processed and aligned using bowtie 1. Each sample was aligned against the rn5 reference genome using bowtie with options “-k 1 -m1”. All gene track and ChIP-seq quantifications were normalized by the number of (uniquely) mapped reads to estimate the “reads per million” (rpm) quantity.
Genome_build: rn5
Supplementary_files_format_and_content: WIG files of ChIP-seq density across the genome
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| Submission date |
May 27, 2020 |
| Last update date |
Apr 25, 2022 |
| Contact name |
Saptarsi Haldar |
| E-mail(s) |
shalda01@amgen.com
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| Organization name |
Amgen
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| Street address |
1120 Veterans Blvd
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL18694 |
| Series (1) |
| GSE151252 |
RNA polymerase II (RNAPII) ChIP-seq analysis evaluating effects of Cdk7 inhibitor THZ1 on responses of neonatal rat ventricular myocytes (NRVM) to adrenergic receptor agonist phenylephrine (PE) |
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| Relations |
| BioSample |
SAMN15031609 |
| SRA |
SRX8404666 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4569626_Pol2PE_control_afterfiting_all.wig.gz |
82.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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