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| Status |
Public on May 07, 2024 |
| Title |
PRO_dTAG_Serum0_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 treated with DMSO or dTAG to induce KAP1 depletion or control.
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Cells were processed for PRO-Seq by washing and permeabilization as described in the methods of the manuscript. The qPRO-Seq protocol was followed with minor modifications (Judd et al. 2020). Cells were seeded in 10 cm tissue culture dishes, treated as indicated and were ~90% confluent prior to collection. For each PRO-Seq, ~4E6 cells were utilized. To prepare permeabilized cells for run-on, cells were first washed twice on the plate with 5 mL of ice-cold 1X PBS. Then, 2.5 mL of ice-cold Cell Permeabilization Buffer (CPB) (10 mM Tris-Cl pH = 8.0 , 250 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA pH = 8.0, 0.1% NP-40, 0.5 mM DTT, 0.05% Tween-20, 0.1% Triton X-100, 10% Glycerol, 1X protease inhibitor, and 2 µL SUPERase-In RNase inhibitor (Thermo, catalog AM2696) per 10 mL was immediately added to the tissue culture dish. Triton X-100 was added to CPB to increase permeabilization percent. Dishes were placed on ice and then immediately scraped with a cell scraper. At this point, cells were collected in a 15 mL tube, placed on ice for 5 min, checked for permeabilization by trypan blue staining (>95%), and then centrifuged in a swinging bucket rotor (1000xg, 4 min, 4°C). Following this, cells were handled using cut tips. The supernatant was removed and samples were resuspended in 1 mL of Cell Wash Buffer (CWB) (10 mM Tris-Cl pH = 8.0, 250 mM Sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA pH = 8.0, 0.5 mM DTT, 10% Glycerol, 1X protease inhibitor, and 2 µL SUPERase-In RNase inhibitor per 10 mL) and centrifuged (1000xg, 4 min, 4°C) for a total of 2 washes. After the second wash, samples were resuspended in 1 mL total Cell Freeze Buffer (CFB) (50 mM Tris-Cl pH = 8.0, 5 mM MgCl2, 0.5 mM DTT, 40% Glycerol, 1.1 mM EDTA pH = 8.0, and 2 µL SUPERase-In RNase inhibitor per 10 mL), counted by hemacytometer, and then spun down in 1.5 mL tubes in an angled rotor centrifuge (1000xg, 5 min, 4°C). Samples were resuspended in 52 μL of CFB for every 4E6 cells, flash frozen in liquid nitrogen, and then frozen at -80°C until ready to use.
Library was prepared with individual reagents following the protocol from Judd et al (dx.doi.org/10.17504/protocols.io.57dg9i6) exactly as described.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
The PRO-seq reads were quality-checked through fastqc/0.11.8 and adapters removed by Cutadapt/2.5.
Trimmed reads were aligned to the hg38 human reference genome using BWA/0.7.5.
Mapped reads were sorted and captured in bam files for further downstream analysis and bigwig files were generated using deeptools.
Assembly: hg38
Supplementary files format and content: BigWig
Library strategy: PRO-Seq
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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 |
| GSE266692 |
KAP1 negatively regulates elongation kinetics to activate signal-induced transcription [PRO-Seq] |
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| Relations |
| BioSample |
SAMN41232147 |
| SRA |
SRX24466466 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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