|
| Status |
Public on May 07, 2024 |
| Title |
dTAG_8hr_Replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
HCT116
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
genotype: KAP1_dTAG_CloneD5
treatment: dTAG_8hr
|
| 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 treated with either DMSO (vehicle control) or dTAG for 8 hr to induce KAP1 depletion.
|
| Extracted molecule |
nuclear RNA |
| Extraction protocol |
For TT-Seq, the protocol from Patrick Cramer’s lab was followed with minor modifications. Three 10 cm tissue culture dishes per condition were seeded such that cells reached 75% confluence 3-days post seeding. On the third day, cells were treated with DMSO or dTAG for 8 hr, after which 500 μM 4-Thiouridine (Sigma, catalog T4509-25MG) was added to the media for 10 min. An extra dish was seeded per condition and ~12E6 cells were counted per plate and thus 36E6 cells/sample were used to prepare RNA. Cells were washed with 5 mL of 1X PBS once and then immediately 1 mL of TRIzol™ Reagent (Thermo, catalog 15596026) was added and followed by 10 min of rocking.
KAPA RNA Hyper+RiboErase HMR kit was used (Roche, catalog 8098131702) following manufacturers instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| 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 and ERCC RNA spike-in mix using star/2.7.3a.
featureCounts (subread/1.6.3) was used to calculate counts across the entire gene of protein coding genes using a .gtf file containing all protein coding genes and ERCC RNA spike-in mix.
EDASeq/2.32.0, RUVSeq/1.32.0, and EdgeR/3.40.1 commands were used for RNA spike-in normalization and differential gene expression analysis of genes that contained at least 10 counts in 6 samples.
Assembly: hg38
Supplementary files format and content: comma-separated txt file with read counts for all features
|
| |
|
| Submission date |
May 06, 2024 |
| Last update date |
May 08, 2024 |
| Contact name |
Ivan D'Orso |
| E-mail(s) |
ivan.dorso@utsouthwestern.edu
|
| Phone |
214-633-1374
|
| Organization name |
UT Southwestern Medical Center
|
| Department |
Microbiology
|
| Lab |
NL3.110A
|
| Street address |
5323 Harry Hines
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-9048 |
| Country |
USA |
| |
|
| Platform ID |
GPL30173 |
| Series (2) |
| GSE246218 |
KAP1 regulates elongation kinetics to activate signal-induced transcription |
| GSE266694 |
KAP1 negatively regulates elongation kinetics to activate signal-induced transcription [TT-Seq] |
|
| Relations |
| BioSample |
SAMN41240544 |
| SRA |
SRX24494004 |
