|
| Status |
Public on Aug 26, 2022 |
| Title |
RNA_Sequencing_siRNA_CPSF3L_TEX_2 |
| Sample type |
SRA |
| |
|
| Source name |
HeLa
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa
treatment: RNA sequencing with siRNA against CPSF3L plus TEX treatment
|
| Treatment protocol |
siRNAs against PCF11 catalogue number 4392420 ID s28363 and s28364; INTS11 siRNA catalogue number 4392420 ID s29893 and s29895; Negative control siRNA catalogue number 4390844). Cells were transfected for using Dharmafect I reagent (Dharmacon) following the manufacturer’s instructions (2 siRNAs per target gene) for 72h. For RNA-seq analysis DNase I-treated, poly(A) selected RNA was treated with Terminator 5′-Phosphate-Dependent Exonuclease (Epicentre; TER51020) according to the manufacturer’s instructions followed enzyme deactivation.
|
| Growth protocol |
HeLa, U2OS, HEK293 and MEF cell lines were grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C. Cell lines were regularly tested for Mycoplasma contamination. Cell lines were authenticated by expression analysis based on RNA-seq. To inhibit RNA polymerase II transcription activity,
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from U2OS and HEK293 cells using QIAzol Reagent (Qiagen; 79306) and Trizol reagent for HeLa cells (15596-018, Ambion life technologies) according to the manufactures protocol followed by DNase I treatment. PolyA selected RNA was isolated using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB; E7490) for U2OS and HEK293 and Oligotex kit (QIAGEN) for HeLa cells and further processed with the NEBNext Ultra Directional RNA Library Prep Kit (NEB; E7420S) for U2OS and HEK293 or SMARTer® Stranded RNA-Seq Kit (Takara; 634839) and illumina Truseq Stranded mRNA Library Prep kit. for HeLa cells or 3′–3′-mRNA-Seq Library Prep Kit (lexogen; 015UG009V0211).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
FASTQ files were aligned to the human hg19 genome using either STAR (STAR_2.5.0a) or TopHat (v.2.0.13) (Kim et al., 2013). Mouse FASTQ files were aligned with the same tools to the mm10 genome. TopHat aligned reads were aligned allowing up to 3 mismatches per read, a maximum gap of 5 bases, and a total edit distance of 8. Peaks were called using mapped reads from all samples, using findPeaks (Homer package, v.3.12)(Heinz et al., 2010) with “-region -size 200 -minDist 250 -strand” parameters. Peaks found within transcripts or up to 5 kb downstream of promoters were included in a GTF file and processed by the DESeq package. Exon-aligned reads were counted using HTSEQ (0.5.4p3). Supplementary_files_format_and_content: TotalRNA_htseq_readcounts.txt is a tab delimited txt file containing raw read counts obtained with htseq over exons of protein coding genes.
|
| |
|
| Submission date |
Mar 29, 2022 |
| Last update date |
Aug 26, 2022 |
| Contact name |
Pierre-Rene Körner |
| Organization name |
Netherlands Cancer Institute (NKI)
|
| Department |
Oncogenomics
|
| Street address |
Plesmanlaan 121
|
| City |
Amsterdam |
| ZIP/Postal code |
1066CX |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE149204 |
Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential |
|
| Relations |
| BioSample |
SAMN27063532 |
| SRA |
SRX14659835 |
