|
| Status |
Public on Jun 20, 2013 |
| Title |
iCLIP2 Puro |
| Sample type |
SRA |
| |
|
| Source name |
HeLa_iCLIP puro
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
treated with: puromycoin
antibody: G-anti-RENT1
antibody vendor: Bethyl
antibody cat. #: A300-038A
|
| Treatment protocol |
For the iCLIP2 Puro experiment, puromycin (Calbiochem) was added to a final concentration of 60.6 mM 6 h before crosslinking.
|
| Growth protocol |
HeLa cells were cultivated in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 100 U/mL Penicillin, 100 mg/mL Streptomycin and 10 % fetal calf serum (FCS) in ten 150 cm2 dishes until 80% confluency (corresponding to a total of 8 x 107 cells).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were subjected to 150 mJ/cm2 UV-C light. After irradiation cells were scrapped off the dishes in PBS. Cell pellets were shock frozen in liquid nitrogen and stored at –80 °C until use. After thawing on ice, cells were lysed in hypotonic gentle lysis buffer for 20 min. The cell lysate was cleared by centrifugation at 4 °C and 13,000 g for 15 min
We then employed the steps of nuclease digestion, primer ligation and blotting of immunoprecipitated complexes to nitrocellulose from the iCLIP method, to minimize cloning of background RNA. Immunoprecipitation (IP) was performed with UPF1-specific antibodies. The cDNA library preparation was performed according to the iCLIP protocol (Konig et al., 2010)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
iCLIP of Upf1 in puromycoin treated HeLa cells
|
| Data processing |
library strategy: iCLIP-seq
For iCLIP samples, first seven nucleotides were trimmed because of bad quality scores prior to mapping to the genome.
Read processing and mapping to the human genome was done with the CLIPZ server (www.clipz.unibas.ch; Khorshid et al., Nucleic Acids Research 2011, 39:D245 (PMID 21087992)).
Genome_build: hg19; UCSC Feb. 2009
Supplementary_files_format_and_content: BED file with uniquely mapped iCLIP sites and uniquely mapped mRNA-seq reads
|
| |
|
| Submission date |
Jun 14, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Andreas R Gruber |
| E-mail(s) |
agruber@tbi.univie.ac.at
|
| Organization name |
University of Basel
|
| Department |
Biozentrum
|
| Lab |
Zavolan
|
| Street address |
Klingelbergstrasse 50-70
|
| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE47976 |
Translation-dependent displacement of UPF1 from coding sequences causes its enrichment in 3’ UTRs |
|
| Relations |
| BioSample |
SAMN02204261 |
| SRA |
SRX306347 |
