|
| Status |
Public on Sep 30, 2024 |
| Title |
MDAMB231_long_siSRSF1_0Gy_bio_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
MDA-MB-231
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
treatment: SRSF1 knockdown, no radiation
|
| Treatment protocol |
4.5 million cells were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific, #13778030) with siRNA against SRSF1 (Dharmacon, #A-018672-13-0005) or a non-targeting siRNA pool (Dharmacon, #D-001910-10-50). One-and-a-half days later, the cells were irradiated in a 137Cesium irradiator at 10 Gy, or mock-irrdiated (0 Gy). Forty-eight hours later, the cells were collected by scraping.
|
| Growth protocol |
Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, #41966029) supplemented with 10% FBS (Sigma-Aldrich, #F9665-500ML)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using RNeasy Mini Kit (QIAGEN)
The library was constructed according to Nanopore protocol, with cDNA and PCR amplification steps. The kit used was PCS111, and the 12 samples were barcoded and ran 5 times (5 flow cells, 12 samples per flow cell) on PromethION.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
| |
|
| Description |
Collected 48 hours after irradiation or mock irradiation
|
| Data processing |
Quality control with Pychopper: pychopper -k PCS111 -r ${report_output} -u ${unclassified_output} -w ${rescued_output} ${input_file} ${full_length_output}
Long-read error correction with short reads (short-read data accession GSE242550) using FMLRC: FM-index Long Read Corrector (FMLRC) v0.1.8 (Mak et al. 2023; Wang et al. 2018b). First, I build a Burrows-Wheeler Transform file from the short reads, as required by FMLRC, using msbwt2-build, then used that to correct the long reads as follows: fmlrc2 "$BWT_PATH" "$input_file" "$output_file". This generated a list of corrected gzipped fasta files for each sample.
aligned those reads to the transcriptome of T2T-CHM13v2.0 with the following command: minimap2 -ax splice rna.fna $file -uf --secondary=no -t 24 > $out_sam_file
Salmon was run in alignment-based mode, with the following command: salmon quant -l SF --ont -t rna.fna -a ${sample}_mapped.sam -o ${sample}_salmon_output
Assembly: human NCBI genome build T2T-CHM13v2.0
Supplementary files format and content: salmon output files (with quant.sf)
|
| |
|
| Submission date |
Jan 04, 2024 |
| Last update date |
Sep 30, 2024 |
| Contact name |
Majd Abdulghani |
| Organization name |
University of Oxford
|
| Street address |
Old Road Campus Research Building
|
| City |
Oxford |
| State/province |
oxfordshire |
| ZIP/Postal code |
OX3 7DQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL26167 |
| Series (1) |
| GSE252534 |
ionizing radiation and SRSF1 knockdown in triple-negative breast cancer cells |
|
| Relations |
| BioSample |
SAMN39257881 |
| SRA |
SRX23097728 |
