|
| Status |
Public on Feb 15, 2024 |
| Title |
Run 1 WT input replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
UOK262
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: UOK262
cell type: renal cell carcinoma
genotype: WT
treatment: input
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were flash frozen by submerging the bottoms of dishes in liquid nitrogen. Cells were collected and lysed by scraping in lysis buffer containing cycloheximide. Cell lysates were triturated and clarified by centrifugation. Input samples were extracted from aliquot of lysate using trizol reagent according to the manufacturer's protocol. Following phase separation, the aqueous phase was applied to a Qiagen RNAeasy column and purified according to kit instructions. For ribosome prototected fragments, lyates were treated with RNase I and loaded onto 15%–45% sucrose gradients. Samples were fractionated and fractions containing 80S monosomes were precipitated with ethanol. After precipitation, monosome containing fractions were resuspended in trizol reagent. Sample workup was performed with a Zymo direct-zol kit following the manufacturer's protocol.
Samples were ribodepleted using Ribopoo ribosome RNA Homo sapiens depletion kit supplemented with custom-made oligos. RPF and input fractions were fragmented and size-selected on denaturing Urea PAGE gels. 3' adaptors sequencing adaptors were added with RNA ligase and reactions were reverse-transcribed with SuperScript IV. Sample cDNA was circularized with circLigase and libraries were PCR amplified to add Illumina sequencing primers. Libraries were sequenced on a NextSeq 2000 P2 instrument.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Data processing |
Read quality analysis was performed with FastQC.Barcode splitting, read collapse, and trimming of adaptor sequences was performed using scripts from the icSHAPE pipeline. Trimmed reads were first mapped to a costume genome that includes rRNA and repeated genomic sequences. Reads that did not map to the costume genome were then aligned to the human genome (hg38) using STAR (v.2.7.6a). Mapped reads were counted by htseq-count program and differential analysis was performed using DEseq2.
Assembly: hg38
Supplementary files format and content: count matrix
Library strategy: Ribo-seq
|
| |
|
| 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 |
GPL30173 |
| Series (2) |
| GSE228561 |
Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma [Ribosome_Profiling] |
| GSE228565 |
Rewiring of RNA methylation by the oncometabolite fumarate in renal cell carcinoma |
|
| Relations |
| BioSample |
SAMN33986950 |
| SRA |
SRX19820072 |
