|
| Status |
Public on Feb 15, 2024 |
| Title |
WT 12 hr replicate 3 |
| Sample type |
SRA |
| |
|
| Source name |
UOK262
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: UOK262
cell type: renal cell carcinoma
genotype: WT
treatment: 4-thiouridine
time: 12 hr
|
| Treatment protocol |
Metabolic labeling to determine RNA half-life followed the method of Herzog et al. Cells were seeded the day before the experiment at approx. 50% confluency. The morning of the experiment, media was removed and replaced with cell media supplemented with 100 µM 4-thiouridine to begin the “pulse”. 4SU-containing media was exchanged every 8 hr during the 24 hr “pulse” period to increase the efficiency of labeling. Following 24 hr labeling, the “pulse” media was removed. Cells were washed 2X with PBS. Cell culture growth medium containing 10 mM uridine (i.e., 100X excess over 4SU) was added to the cells to begin the “chase”.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At select time points of interest, “chase” media was removed, and cells were lysed in using Trizol Reagent according to the manufacture’s protocol with the following modifications: RNA isolation was performed in the dark and DTT was added to the aqueous phase to keep samples under reducing conditions.
Library preparation was performed using QuantSeq 3' mRNA library prep kit for Illumina using the manufacturer’s protocol and recommended maximum input amount
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Read quality analysis was performed with FastQC. Following the recommendation of Herzog et al., read 2 was discarded. Demultiplexed fastq files were applied to the SLAMDUNK pipeline (v/0.4.2) and mapped against hg38. The optional --multimappers flag was enabled. Following the pipeline, the output count file was collapsed used the included SLAMDUNK Alleyoop collapse module. Curve fitting was performed according to a first-order decay reaction in R (v3.6.3)
SLAMDUNK pipeline (v 0.4.2)
Assembly: hg38
Supplementary files format and content: tsv count file
|
| |
|
| Submission date |
Mar 30, 2023 |
| Last update date |
Feb 15, 2024 |
| Contact name |
Pedro J Batista |
| E-mail(s) |
pedro.batista@nih.gov
|
| Phone |
3014356294
|
| Organization name |
National Institutes of Health
|
| Department |
National Cancer Institute
|
| Lab |
Cell Biology
|
| Street address |
37 Convent Street Bldg 37
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL21290 |
| Series (2) |
| GSE228559 |
Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma [SLAM-seq] |
| GSE228565 |
Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma |
|
| Relations |
| BioSample |
SAMN33986873 |
| SRA |
SRX19820010 |
