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| Status |
Public on Jan 06, 2023 |
| Title |
LP-1_DMSO_4h_RNAPIISer2P_ChIP-seq |
| Sample type |
SRA |
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| Source name |
LP-1_DMSO_4h_RNAPIISer2P
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| Organism |
Homo sapiens |
| Characteristics |
cell line: LP-1
cell type: multiple myeloma
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| Treatment protocol |
LP-1 cells were treated with DMSO or 0.5 μM JQ1 for 4 hours. Cells were fixed with 1% formaldehyde by the addition of 37% formaldehyde directly to the culture medium. Cells were fixed for 10 minutes at room temperature (RT) and quenched with 0.125 M glycine, pH 7.9.
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| Growth protocol |
LP-1 cells were cultured at 37 ˚C under 5% CO2 in IMDM medium supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100 µg/mL streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were incubated in lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.2 % NP-40) for 1 hour at 4 ˚C. Chromatin was digested at a concentration of 25 x106 cells/mL with 350 U/mL MNase (Roche,10107921001) for 20 minutes at RT in MNase reaction buffer (55 mM Tris-HCl pH 7.5, 0.05 mM EDTA, 2.5 mM MgAc2, 12.5 % glycerol, 25 mM KCl, 4 mM MgCl2, 1 mM CaCl2). The digestion was stopped with 10 mM EDTA. The chromatin was then sonicated with a 550 Sonic Dismembrator (Fisher Scientific) on power 4, using cycles of 0.7 seconds on and 1.4 seconds off for a total processing time of 3 minutes. Lysates were centrifuged at 21,000 x g for 10 min at 4 ˚C. 5 x106 cell equivalents were used for each ChIP assay, diluted to 1 mL with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 1.2 mM EDTA, 1.1% Triton-X 100, 167 mM NaCl, 0.01 % SDS). Chromatin was pre-cleared for 2 hours at 4 ˚C with Protein A Dynabeads (Life Technologies,10001D) in blocking buffer (1x PBS, 0.5 % BSA, 0.1% Tween-20). Beads were captured and pre-cleared chromatin was incubated with antibodies overnight at 4 ˚C. Samples were then incubated with Protein A Dynabeads in blocking buffer for 2 hours at 4 ˚C. Beads were washed 3x with RIPA buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate, 0.1 % SDS) and 1x with RIPA high salt buffer (10 mM Tris-HCl pH 8.0, 360 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate, 0.1 % SDS). Bound chromatin was eluted with elution buffer (10 mM Tris-HCl, 1 % SDS, 300 mM NaCl, 10 mM EDTA pH 8.0). Crosslinks were reversed by incubation at 65 ˚C for 6 hours. Eluted chromatin was treated with 150 U/mL RNase A for 30 minutes at 37 ˚C and digested with 75 µg/mL proteinase K for 1 hour at 55 ˚C. ChIP DNA was purified with using a MinElute PCR Purification Kit (QIAGEN) and quantified with a Qubit fluorimeter (Life Technologies).
Library DNA fragments for BRD4, RNAPII, RNAPIISer2P ChIP and input DNA were ligated to Illumina adapters and size selected (200-250 bp) on a 2% agarose gel and PCR amplified for 18 cycles. Libraries of H3K27ac ChIP and input DNA were constructed using an Ovation Ultralow DR Multiplex System Kit (NuGEN) using 15 cycles of amplification. Libraries were size selected (250-300 bp) on a 2% agarose gel.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
ChIP antibody: RNAPIISer2P (Abcam, ab5905)
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| Data processing |
Reads from ChIP-seq were mapped to the hg19 version of the human genome using BWA version 0.7.10. Duplicate reads were removed using samtools’ sort and rmdup functions.
IGVTools count was run with an extension of 100 and a window size of 25 bp to generate coverage signal in WIG format. The WIG files were scaled to a mean of 1.0 assuming an effective genome size of 2,700,000,000 bases.
Genome_build: hg19
Supplementary_files_format_and_content: bed file with enhancers, tab-delimited text file including BRD4 ChIP-seq signal in enhancers and promoters and wig files
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| Submission date |
Jul 01, 2015 |
| Last update date |
Jan 06, 2023 |
| Contact name |
Barbara M Bryant |
| E-mail(s) |
barbara.bryant@constellationpharma.com
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| Organization name |
Constellation Pharmaceuticals
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| Street address |
215 First Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (2) |
| GSE70448 |
BRD4 loading at the transcription start site mediates pause-release and underlies the disproportionate transcriptional response to BET bromodomain inhibition [ChIP-Seq] |
| GSE70450 |
BRD4 loading at the transcription start site mediates pause-release and underlies the disproportionate transcriptional response to BET bromodomain inhibition |
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| Relations |
| BioSample |
SAMN03835675 |
| SRA |
SRX1078946 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1755003_07_LP1_cont_Pol2Ser2.wig.gz |
282.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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