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| Status |
Public on May 07, 2024 |
| Title |
PolII_DMSO_Serum30_Replicate1 |
| Sample type |
SRA |
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| Source name |
HCT116
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
genotype: KAP1_dTAG_CloneD5
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| Growth protocol |
All cell lines were maintained in DMEM supplemented with 7% fetal bovine serum (FBS) and antibiotics (Pen/Strep) in humidified atmosphere with 5% CO2 at 37C. For treatments, cells were placed in serum-free media for 48 hr prior to serum treatment for the corresponding time point. 8 hr prior to serum treatment, cells were pre-incubated with DMSO (vehicle control) or dTAG to induce KAP1 depletion.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each ChIP, 1 x 15-cm tissue culture dish (yielding ~30E6 cells/plate) was utilized and seeded according to the experimental design in each figure (DMSO/dTAG +/- serum at different time points), and 20E6 cells were utilized per ChIP. For all Pol II ChIPs, cells were crosslinked with 0.5% methanol-free formaldehyde (Thermo Fisher Scientific, 28908) by adding directly to the dish in media at room temperature for 10 min with rocking and neutralization with 150 mM glycine for 5 min with rocking. For the ChIPs including HA (KAP1), SPT5, CDK9, TFIIB, MED1, and CDK7, 1% formaldehyde for 10 min was used instead of 0.5%. For all experiments, the media was removed and cells were washed twice with cold 1X PBS, cold PBS was added and then cells were scraped off the plate. Cells were centrifuged (1000xg, 5 min, 4°C), and pelleted, flash frozen in liquid nitrogen, and frozen at -80°C until ready to use. To perform the ChIP, cells were resuspended in 2 mL/dish of Farnham Lysis Buffer (5 mM PIPES pH = 8.0, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, 1X Protease Inhibitor (RPI, catalog P50900-1)), counted by hemacytometer, resuspended to 10E6 cells/mL, nutated for 30 min at 4°C, and then centrifuged to isolate nuclei (1000xg, 5 min, 4°C). The supernatant was removed and nuclei resuspended in Szak’s RIPA Buffer (50 mM Tris-HCl pH = 8.0, 1% NP-40, 150 mM NaCl, 0.5% Na-Deoxycholate, 0.1% SDS, 2.5 mM EDTA pH = 8.0, 1 mM PMSF, and 1X Protease Inhibitor) at a concentration of 25E6 nuclei/mL.
Libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems, catalog KK8502) following the manufacturers protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Description |
CPM_RPB3_DMSO_S30_Merged.bw
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| Data processing |
raw fastq data files were ran through fastqc/0.11.8 and low quality reads/adapter contamination were removed using trimgalore/0.6.4
Reads were mapped to the hg38 human reference genome using bowtie2/2.4.2. Reads were also mapped to the dm6 Drosophila genome to extract reads originating from the spike-ins chromatin for normalization purposes.
Duplicates were marked and removed using picard/2.10.3 and then files were sorted and indexed using samtools/1.6 in preparation for Bigwig generation.
Normalized BigWigs were made from sorted bam files using Deeptools command bamCoverage using either CPM normalization or scale factors generated from sorted Drosophila bam files.
Assembly: hg38
Supplementary files format and content: bigWig
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| Submission date |
May 06, 2024 |
| Last update date |
May 07, 2024 |
| 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 |
GPL30173 |
| Series (2) |
| GSE246218 |
KAP1 regulates elongation kinetics to activate signal-induced transcription |
| GSE266691 |
KAP1 negatively regulates elongation kinetics to activate signal-induced transcription [ChIP-Seq] |
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| Relations |
| BioSample |
SAMN41237780 |
| SRA |
SRX24475624 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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