|
| Status |
Public on Jan 28, 2022 |
| Title |
RNA-Seq of HEK293 cells after knockdown of SP1, replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Human embryonic kidney cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
knockdown: siSP1
|
| Treatment protocol |
Cells were transfected with siSP1 Silencer Select siRNA(Thermo Fisher) using Lipofectamine RNAiMAX (Thermo Fisher), as recommended by the manufacturer. Silencer Select control siRNA (Thermo Fisher) was used as a control (siMock). Cells were harvested 72 hours post transfection.
|
| Growth protocol |
HEK293 cells were grown in DMEM supplemented with 10% FBS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin, and maintained at 37°C with 5% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated from lysates using Trizol (Thermo Fisher Scientific) as described by the manufacturer and was treated with DNAse (Thermo Fisher).
1000 ng per sample was used in the library preparation. Sequencing libraries were constructed using Illumina’s TruSeq Stranded mRNA sample preparation protocol, as described in Illumina’s TruSeq mRNA sample preparation guide.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Base calls were processed with bcl2fastq2 (Illumina) version 2.18.
For differential mRNA expression analysis, gene-level read counts were quantified using Salmon (version 0.8.2) and normalized by variance-stabilizing transformation using DESeq2.
To estimate the differential poly(A) site usage, we used QAPA with default settings, essentially as previously described (Ha KCH et. al. 2018. Genome Biology).To quantify the relative usage of proximal poly(A) sites, QAPA calculates the percentage of expression of a single 3′ UTR over the total expression level of all 3′ UTRs for a given gene. Lengthening events were defined as those with percentage of proximal Poly(A) Usage Index (PAU) group difference between siSp1 and siMock (PAU_Group_diff) < −20% and shortening events with PAU_Group_diff > 20%.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files for Poly(A) usage (PAU) from QAPA analysis, PAU difference between siSP1 and siMock from APA analysis, and differential gene expression from DESeq2 analysis
|
| |
|
| Submission date |
Jan 29, 2021 |
| Last update date |
Jan 28, 2022 |
| Contact name |
Ulrich Braunschweig |
| Organization name |
University of Toronto
|
| Department |
Donnelly Centre
|
| Lab |
Benjamin J. Blencowe
|
| Street address |
160 College Street
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE165771 |
Regulation of alternative polyadenylation by the C2H2-zinc finger protein Sp1 [RNA-Seq] |
| GSE165772 |
Regulation of alternative polyadenylation by the C2H2-zinc finger protein Sp1 |
|
| Relations |
| BioSample |
SAMN17674844 |
| SRA |
SRX9975283 |
