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| Status |
Public on Oct 26, 2018 |
| Title |
PolII_468_5uM_6h_293T_rep1 |
| Sample type |
SRA |
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| Source name |
293T
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human embryonic kidney cells
drug treatment: KL-2
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| Treatment protocol |
Treatment withKL-1 or KL-2 inhibitors was administrated only once
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| Growth protocol |
HEK293T (ATCC), HCT116 (ATCC), Flag-AFF1 and Flag-AFF4 stable cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, catalog No. F6178, Sigma). NCI-H2171 [H2171] (ATCC CRL-5929) and SW1271 (ATCC CRL-2177) small cell lung cancer cells were maintained in RPMI-1640 and DMEM/F12 medium supplemented with 20% FBS. The Jurkat J-Lat full-length cells (6.3) (Cat # 9846) were provided by the NIH AIDS Reagent Program and cultured in RPMI-1640 medium with 10% FBS. Drosophila S2 cells were maintained in Schneider's medium. The wild-type Pol II, slow Pol II (R749H) and fast Pol II (E1126G) mutant HEK293T cell lines (Fong et al., 2014) were provided by Dr. David Bentley (University of Colorado School of Medicine) and cultured in DMEM with 10% FBS. After induction with doxycycline (2.0 μg/mL) for 16 hr, speed-mutant cells were treated with α-amanitin (2.5 μg/mL, Sigma) for 42 hr prior to ChIP-seq analysis.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq was done according to a previously published protocol (Liang et al., 2015). Briefly, cells were crosslinked with 1% paraformaldehyde for 10 min and were quenched with glycine for 5 min at room temperature. Fixed chromatin was sonicated with a Covaris Focused-ultrasonicator for 6 minutes and immunoprecipitated with the indicated antibody. DNA after ChIP protocol was isolated using QIAGEN PCR purification kit.
ChIP-sequencing libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP-seq PolII
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| Data processing |
ChIP-seq reads were aligned to the Drosophila genome (UCSC dm3) or human genome (UCSC hg19). Alignments were processed with Bowtie version 1.1.2, allowing only uniquely mapping reads with up to two mismatches within the 50 bp read. The resulting reads were extended to 150 bases toward the interior of the sequenced fragment and normalized to total reads aligned (reads per million, rpm). Peaks were called using MACS (model based analysis of ChIP-Seq) (Zhang et al., 2008) version 1.4.2 using default parameters. Ensembl version 75 transcripts were chosen with the highest total coverage from the annotated TSS to 200 nt downstream for protein coding genes that also have a refseq identifier, were at least 2 kb long, and 2 kb away from the nearest gene. For pausing indexes, the promoter region was defined as -200 bp upstream to 400 bp downstream, and the body region was the remainder of the entire gene body. The ratio of the average coverage (r.p.m) of the promoter over the average coverage of the gene body was then taken to be the Pausing Index. ECDF plots were made in R version 3.3.3 using the ecdf function. P-values were calculated with a one-sided Kolmogorov-Smirnov test. Heatmaps tables were made for the indicated windows around the TSS or TES using the average coverage (r.p.m) in 25bp & 50bp bins (50bp bins for 50kb downstream of the TSS). Meta gene tables were made by approximating the coverage across all genes to the same length. All ChIP-seq heatmaps were sorted by the decreasing coverage in indicated windows by the control sample and visualized using JavaTreeView version 1.6.4 (Saldanha, 2004). Average profile plots were made by averaging the coverage for all genes using colMeans in R.
Genome_build: hg19
Supplementary_files_format_and_content: Library normalized bigWig files were provided for all samples
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| Submission date |
Aug 29, 2018 |
| Last update date |
Oct 28, 2018 |
| Contact name |
Ali Shilatifard |
| E-mail(s) |
[email protected]
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| Organization name |
Northwestern University Feinberg School of Medicine
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| Department |
Department of Biochemistry and Molecular Genetics
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| Lab |
Shilatifard Lab
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| Street address |
320 E Superior St
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE112608 |
Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy |
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| Relations |
| BioSample |
SAMN09932794 |
| SRA |
SRX4620890 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3360538_PolII_468_5uM_6h_293T_rep1.bw |
201.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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