|
| Status |
Public on Oct 01, 2019 |
| Title |
DMSO_TNF1 |
| Sample type |
SRA |
| |
|
| Source name |
HeLa cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa cells
treatment: DMS + TNF
|
| Treatment protocol |
HeLa cells were metabolically labeled with 4-thio-uridine (4sU) for 2 hours in the presence of DMSO or SPI-21 and in the presence or absence of TNFα.
|
| Growth protocol |
HeLa cells were grown and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Gibco) and 1% penicillin-streptomycin. To avoid basal NF-кB activity, HeLa cells were kept from reaching confluence and re-plated after initial thawing no more than 10 times.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
4sU-labeled RNA was biotinylated, purified and captured using Streptavidin-coated magnetic beads.
4sU-seq RNA samples were reverse transcribed using inhouse mRNA Seq protocol w/o PolyA capture with random hexamers.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
batch1
cellular labeled RNA
|
| Data processing |
Library strategy: 4sU-seq
Sequences were analysed using the RNA-Seq pipeline of UTAP (Kohen et al. BMC Bioinformatics (2019) 20:154 https://0-doi-org.brum.beds.ac.uk/10.1186/s12859-019-2728-2) with batch correction.
In this pipeline the reads were trimmed using cutadapt (DOI: 10.14806/ej.17.1.200) and mapped to genome hg38 using STAR (DOI: 10.1093/bioinformatics/bts635) v2.4.2a (default parameters).
The pipeline quantifies the genes annotated in RefSeq (downloaded from iGenomes).
Gene expression counting was done using STAR.
Further analysis is done for genes having minimum 5 read in at least one sample.
Normalization of the counts and differential expression analysis was performed using DESeq2 (DOI: 10.1186/s13059-014-0550-8) with the parameters: betaPrior=True, cooksCutoff=FALSE, independentFiltering=FALSE. Raw P values were adjusted for multiple testing using the procedure of Benjamini and Hochberg. In the contrast DMSO_TNF_vs_DMSO and SPI21_TNF_vs_SPI21 fdrtools was applied. Batch corrected normalized log2 counts are termed combat values and were calculated using “sva” package of R
The thresholds for significant differential expression (DE) are: padj <= 0.05 & |log2FoldChange| >= 1 & baseMean >= 5
Genome_build: hg38
Supplementary_files_format_and_content: Excel
|
| |
|
| Submission date |
Aug 19, 2019 |
| Last update date |
Oct 01, 2019 |
| Contact name |
Dena Leshkowitz |
| E-mail(s) |
dena.leshkowitz@weizmann.ac.il
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Bioinformatics Unit, Life Sciences Core Facilities
|
| Street address |
P.O.B. 26
|
| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE136025 |
Genome-wide view of the impact of Spt5-Pol II inhibitors (SPIs) on transcription [4sU-seq] |
| GSE136026 |
Targeting Spt5-Pol II small-molecule inhibitors uncouple distinct activities and reveal additional regulatory roles |
|
| Relations |
| BioSample |
SAMN12604243 |
| SRA |
SRX6743175 |
