|
| Status |
Public on Dec 13, 2012 |
| Title |
Immunoprecipitate Replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
FlpIn T-Rex Human embryonic kidney 293 stable cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: FlpIn T-Rex Human embryonic kidney 293 stable cell line
rip antibody: anti-FLAG M2 [Sigma-Aldrich, cat# F1804, lot#101M6216]
|
| Treatment protocol |
Expression of stable transgenes were induced by incubation with 1 µg/ml doxycyline for 16 h. Cells were then lysed in NP-40 lysis buffer (50 mM HEPES-KOH at pH 7.4, 150 mM KCl, 2 mM EDTA, 0.5 mM DTT, 1 mM NaF, and 0.5% NP-40, EDTA-free protease inhibitor cocktail (Roche)), and centrifuge-clarified supernatants were collected. Total RNA was collected prior to IP and labelled as Lysate. Supernatants were subjected to IP using anti-FLAG M2 monoclonal antibodies conjugated to Protein G-ferromagnetic beads (Dynabeads, Invitrogen). Immunoprecipitates were washed three times using NP-40 lysis buffer (containing 300 mM KCl).
|
| Growth protocol |
FlpIn T-Rex HEK293 stable cell lines were cultured in DMEM (11995, Invitrogen) supplemented with 10% Fetal bovine serum, 2 mM L-Glutamine, 50 units of Penicillin, 50 µg/ml Streptomycin, 100 µg/ml Hygromycin, and 15 µg/ml Blasticidin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total or immunoprecipitated RNA was extracted by phenol-chloroform, followed by ethanol precipitation. Rneasy Mini Kit (Invitrogen) was used according to manufacturer's instructions as a final step.
|
| 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 |
IP_2
RNA from immunoprecipitated FlagHA FMRP
|
| Data processing |
R package 'affy' for normalization with the following normalization parameters: 'expresso(fmrRIP, normalize.method=quantiles.robust, bgcorrect.method=rma,pmcorrect.method=pmonly, summary.method=mas)'
|
| |
|
| Submission date |
Jul 19, 2012 |
| Last update date |
Dec 13, 2012 |
| Contact name |
Manuel Ascano, Jr. |
| Organization name |
The Rockefeller University
|
| Department |
HHMI/Laboratory of RNA Molecular Biology
|
| Lab |
Tuschl Laboratory
|
| Street address |
1230 York Avenue Box 186
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021-6307 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE39504 |
FMR1 targets distinct mRNA sequence elements to regulate protein expression [Affymetrix] |
| GSE39686 |
FMR1 targets distinct mRNA sequence elements to regulate protein expression |
|
