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Status |
Public on Dec 13, 2012 |
Title |
Lysate Replicate 2 |
Sample type |
RNA |
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|
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: n/a
|
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).
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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).
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|
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Hybridization protocol |
3 µg cRNA hybridized to HGU133 Plus 2.0 Arrays for 16 h at 45ºC
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Scan protocol |
Genechips scanned on Affymetrix Genechip 300 7G scanner
|
Description |
Lys_2
|
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)'
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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 |
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