|
| Status |
Public on Apr 11, 2014 |
| Title |
4suRNA_MOV10siRNA |
| Sample type |
SRA |
| |
|
| Source name |
HEK293 cell culture
|
| Organism |
Homo sapiens |
| Characteristics |
purification: oligo(dT) beads
cell line: HEK293
transfection: MOV10 siRNA
|
| Treatment protocol |
For global mRNA for half-life measurements, mock- or MOV10 siRNA transfected HEK293 cells were labeled with 700 µM 4SU for 60 min.
|
| Growth protocol |
HEK293 were grown in D-MEM high glucose with 10% (v/v) fetal bovine serum, 1% (v/v) 2 mM L-glutamine, 1% (v/v) 10,000 U/ml penicillin/10,000 µg/ml streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the miRNeasy kit (Qiagen). 4SU residues were biotinylated using EZ-Link biotin-HPDP (Thermo Fisher Scientific). Biotinylated 4SU-labled RNA was separated from non-labeled RNA using μMACS Streptavidin MicroBeads (Miltenyi) and 4SU-labeled RNA was eluted from μColumns by addition of 100 mM DTT. RNA was recovered from the flow-though and 4SU-labeled fractions using MinElute Spin columns (Qiagen). Input (total), flow-though (non-labeled RNA) and eluted (4SU-labled RNA) samples were used for poly(A)+ mRNA library preparation following the TruSeq RNA sample Prep v2 LS protocol (Illumina). The libraries were sequenced on an Illumina HiSeq2500 for 100 cycles (multiplexed 1× 101 + 7 index). mRNA half-lives were computed as previously described (Schwanhausser et al., 2011)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Read demultiplexing, read filtering and adapter trimming was done using flexbar (Dodt et al, 2012).
We used TopHat2 (version 2.06) (Trapnell et al., 2009 and Kim et al. 2013) for spliced alignment of reads to the human reference genome sequence (hg18).
mRNA half-lives were determined as described in Schwanhaeusser et al. Nature 2011 from the three fractions total/4suRNA/flowthrough
Genome_build: hg18
|
| |
|
| Submission date |
Apr 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Markus Landthaler |
| E-mail(s) |
markus.landthaler@mdc-berlin.de
|
| Phone |
+49-30-9406-3026
|
| Organization name |
Max-Delbrück-Center for Molecular Medicine
|
| Department |
Berlin Institute for Medical Systems Biology
|
| Street address |
Robert-Rössle-Straße 10
|
| City |
Berlin |
| ZIP/Postal code |
13125 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE56748 |
MOV10 Is a 5' to 3' RNA Helicase Contributing to UPF1 mRNA Target Degradation by Translocation along 3'UTRs (expression) |
| GSE56751 |
MOV10 Is a 5' to 3' RNA Helicase Contributing to UPF1 mRNA Target Degradation by Translocation along 3'UTRs |
|
| Relations |
| BioSample |
SAMN02726093 |
| SRA |
SRX515682 |
