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| Status |
Public on Oct 09, 2015 |
| Title |
Total_Pol_II_ChIP-Seq |
| Sample type |
SRA |
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| Source name |
Hct 116 Cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell type: Colonic Endothelial Cells
chip antibody: Pol II (N-20) (SCBT; sc-899X)
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| Growth protocol |
Hct 116 cells were cultured in Dulbecco's modified Eagles's Medium (DMEM) supplemnted with 10% fetal bovine serum (FBS) at 37C with 5% CO2 (HyClone).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature in culture media, and the reaction was stopped by the addition of glycine (125 mM final). Cells were washed twice with PBS and re-suspended in 1 ml of nuclei extraction buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, protease inhibitor (Roche)) at 2x107 cells/ml and incubated for 10 min at 4°C. Cell nuclei were collected by centrifugation at 3000 rpm for 5 min at 4°C, and re-suspended in Szak's RIPA buffer (50 mM Tris-HCl pH 8.0, 1% NP-40, 150 mM NaCl, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 0.5 mM PMSF, protease inhibitor) at 5x107 nuclei/ml. Nuclear pellets were sonicated using the Bioruptor water bath sonicator (Diagenode) using the high setting with 30 sec on 30 sec off for 50 minutes to generate 100-300 bp DNA fragments. Approximately 25-50x106 nuclei were used for each ChIP. For the ChIP step, 2-4 ug of antibody was conjugated with 40 uL of protein G dynabeads (Invitrogen) and blocked with 0.16% bovine serum albumin for 1 hour at 4°C. Antibody-conjugated dynabeads were incubated with sheared chromatin suspension for 2 hr at 4°C. Then the beads were sequentially washed sequentially two times with the following buffers: Szak's RIPA buffer (see above), low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton, 0.1% SDS, 2 mM EDTA), LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 20 mM Tris, pH 8.0), and 1 mM Tris-EDTA pH 8.0. Chromatin immunocomplexes were eluted by incubation for 10 min at 65°C with 1% SDS and 100 mM NaHCO3, and cross-linking was reversed by incubation in the solution adjusted to 200 mM NaCl and proteinase K (20 μg) for 1 hr at 65°C. DNA was then purified using the Zymo ChIP DNA Clean & Concentrator Kit (Zymo Research). Final ChIP DNA was quantified on a Qubit 2.0 Fluorometer (Invitrogen) and approximately 20 ng of DNA were submitted for library preparation.
ChIP DNA was submitted to McDermott Center Sequencing Core at UT Southwestern Medical Center for library preparation and high-throughput sequencing. Samples were end repaired, 3’-end adenylated and barcoded with multiplex adapters (Applied Biosystems) and samples were PCR amplified (~14 cycles) and size selected with Ampure Beads. Sequencing was done using the Illumina NextSeq 500 using 50 bp read length.
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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 data set for Total Pol II
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| Data processing |
ChIP-Seq reads were aligned to the reference genome hg19 using bowtie v1.1. The flag "-m 1" was used
Peaks were called using macs2 using the default fdr threshold of p-value < 0.05
BedGraph tracks were generated using macs2 and then converted to BigWig using bedGraphToBigWig
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-Seq: Tab-delimited peak files generated by macs2
Supplementary_files_format_and_content: BigWig track files containing aligned read densities
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| Submission date |
Sep 02, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ivan D'Orso |
| E-mail(s) |
ivan.dorso@utsouthwestern.edu
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| Phone |
214-633-1374
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| Organization name |
UT Southwestern Medical Center
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| Department |
Microbiology
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| Lab |
NL3.110A
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| Street address |
5323 Harry Hines
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-9048 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE72622 |
KAP1 Recruitment of the 7SK snRNP to Promoters Enables Transcription Elongation by RNA Polymerase II |
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| Relations |
| BioSample |
SAMN04026662 |
| SRA |
SRX1177901 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1866693_Pol-II-Chip.MACS2_peaks.narrowPeak.gz |
2.0 Mb |
(ftp)(http) |
NARROWPEAK |
| GSM1866693_Pol-II-Chip.bigWig |
156.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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