|
| Status |
Public on Jun 09, 2014 |
| Title |
18.KD.t25 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T-rex cells, 18.KD.t25
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T-rex
cell line clone: 18
recql5 status: knock-down
treatment: Doxycycline 25 min
|
| Treatment protocol |
Cells were treated with 100mM of DRB to stop transcription, after a double wash with PBS, cells were released in fresh medium and 10', 25' and 40' after release cells were processed for Run-On experiments.
|
| Growth protocol |
Constitutively KD cells were grown in presence of Doxycycline to induce physiological level of an shRNA resistant full length version of RECQL5, or in the absence of Doxycycline to shut off the expression of the protein for 4 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Transcription competent nuclei were prepared using the Nuclei Isolation Kit (Sigma) according to manufacturers' recommendations and Run-On was performed as previously described (Core et al., 2008)
Strand specific RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.8.2
GRO-seq reads were aligned to the hg19 genome assembly using BWA v0.5.9 with the following configurations: -l 36, -n 3
BAM files representing the same sample were merged and reads mapping to Ensembl rRNAs or to regions blacklisted for mappability by the Encode project were removed.
Further analysis was conducted using Bioconductor. Reads were extended to 250bp and each sample was normalised to a read depth of 20 million. A subset of the protein coding human Ensembl transcriptome was created by filtering for transcripts >=30kb. The largest transcript was selected per gene, resulting in a list of 8529. Bp level coverage of the region 2kb upstream to 120kb downstream of each transcript's TSS was calculated for each sample.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files containing mean normalized read depth across selected protein coding Ensembl transcripts. Covers the region -2kb upstream to +120kb downstream of the TSS.
|
| |
|
| Submission date |
Jul 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard James Mitter |
| Organization name |
The Francis Crick Institute
|
| Department |
Bioinformatics & Biostatistics
|
| Street address |
1 Midland Road
|
| City |
London |
| State/province |
LONDON |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE49133 |
Mapping of nascent RNA upon release of DRB in WT and KD of RECQL5 |
| GSE49134 |
RECQL5 Controls Transcript Elongation and Suppresses Transcription-Associated Genome Instability |
|
| Relations |
| BioSample |
SAMN02262045 |
| SRA |
SRX327273 |
