|
| Status |
Public on Feb 26, 2013 |
| Title |
LIN28B_knockdown_rep1 [Affymetrix] |
| Sample type |
RNA |
| |
|
| Source name |
HEK 293 cells inducibly expressing FLAG/HA tagged LIN28B; transgene not induced; siRNA transfected; replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
|
| Treatment protocol |
siRNA transfections of HEK293 T-REx Flp-In cells were performed in 6-well format using Lipofectamine RNAiMAX (Invitrogen) as described by the manufacturer. Detailed experimental procedure can be found in the Methods section of Hafner et al., RNA, in press Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition
|
| Growth protocol |
HEK293 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-LIN28B was induced by addition of 250 ng/ml doxycycline 15 to 20 h before crosslinking. Detailed experimental procedure can be found in the Methods section of Hafner et al., RNA, in press, Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition
|
| 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 Array (Affymetrix).
|
| |
|
| Hybridization protocol |
3 µg cRNA hybridized to HGU133 Plus 2.0 Arrays for 16 h at 45ºC
|
| Scan protocol |
Genechips scanned on Affymetrix Genechip 300 7G scanner
|
| Description |
Gene expression data from HEK293 cells inducibly expressing FLAG/HA tagged LIN28B after knockdown with siRNA
|
| 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 procedure can be found in the Supplement to Hafner et al. Cell, 2010,141(1),129-141 Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP
|
| |
|
| Submission date |
Feb 25, 2013 |
| Last update date |
Feb 26, 2013 |
| Contact name |
Miguel Brown |
| E-mail(s) |
miguel.a.brown@gmail.com
|
| Organization name |
The Rockefeller University
|
| Street address |
1230 York Ave Box 186
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE44613 |
Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition [Affymetrix] |
| GSE44616 |
Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition |
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