|
| Status |
Public on Apr 29, 2010 |
| Title |
HEK 293 cells expressing FLAG/HA-tagged IGF2BP1, siRNA-transfected, replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
HEK 293, IGF2BP siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK 293
sirna: IGF2BP1, IGF2BP2, IGF2BP3
|
| Treatment protocol |
siRNA, miRNA and 2'-O-methyl oligonucleotide transfections of HEK 293 T-REx Flp-In cells were performed in 6-well format using Lipofectamine RNAiMAX (Invitrogen) as described by the manufacturer. Detailed experimental procedures can be found in the Supplement to PMID 20371350.
|
| Growth protocol |
HEK 293 T-REx Flp-In cells (Invitrogen) 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, 100 µg/ml zeocin and 15 µg/ml blasticidin. Cell lines stably expressing FLAG/HA-tagged proteins were generated by co-transfection of pFRT/TO/FLAG/HA or pFRT/FLAG/HA constructs with pOG44 (Invitrogen). Cells were selected by exchanging zeocin with 100 µg/ml hygromycin. Expression of FLAG/HA-IGF2BP1, -2, -3 and TNRC6A, B and C was induced by addition of 250 ng/ml doxycycline 15 to 20 h before crosslinking. Detailed experimental procedures can be found in the Supplement to PMID 20371350.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
2 µg of purified total RNA was used in the One-Cycle Eukaryotic Target Labeling Assay (Affymetrix) according to manufacturer's protocol. Biotinylated cRNA targets were cleaned up, fragmented, and hybridized to Human Genome U133 Plus 2.0 Arrays (Affymetrix).
|
| |
|
| Hybridization protocol |
3 µg cRNA was hybridized to HGU133 Plus 2.0 Arrays for 16 h at 45ºC.
|
| Scan protocol |
GeneChips were scanned on an Affymetrix GeneChip 3000 7G scanner.
|
| Description |
Gene expression data from HEK 293 cells inducibly expressing FLAG/HA-tagged IGF2BP1 after knockdown with IGF2BP1-3 siRNA.
IGF2BP siRNA 2
|
| Data processing |
We imported the CEL files into the R software (http://www.R-project.org) using the BioConductor affy package (Gentleman et al., 2004). The transcript probe set intensities were background-corrected, adjusted for non-specific binding and quantile normalized with the GCRMA algorithm (Wu, 2006). Probe sets with more than 6 of the 11 probes mapping ambiguously to the genome were discarded, as were probe sets that mapped to multiple genes. We then collected all probe sets matching a given gene, and we selected for further analysis the RefSeq transcript with median 3'UTR length corresponding to that gene. In total, 16,063 transcripts were identified. The log-intensity of probe sets mapping to the gene were then averaged to obtain the expression level per RefSeq transcript. The changes of transcript abundances were computed as the logarithm of the ratio of transcript expression in the cocktails of siRNA-treated samples and mock-transfected cells. Detailed analysis procedures can be found in the Supplement to PMID 20371350.
|
| |
|
| Submission date |
Apr 28, 2010 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Markus Hafner |
| E-mail(s) |
mhafner@rockefeller.edu
|
| Phone |
+1 212 327 7696
|
| Fax |
+1212 327 7652
|
| Organization name |
The Rockefeller University
|
| Department |
Laboratory of RNA Molecular Biology
|
| Lab |
Tuschl lab
|
| Street address |
1230 York Ave, Box 186
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE21575 |
Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: IGF2BP data |
| GSE21578 |
Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP |
|
| Relations |
| Reanalyzed by |
GSE119087 |
