|
| Status |
Public on Mar 28, 2016 |
| Title |
RBFOX2 knockdown shRNA2 Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
RBFOX2 knockdown shRNA2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: 293T
protocol: shRNA TRCN0000074546
genotype/variation: RBFOX2 knockdown
|
| Treatment protocol |
Knockdowns were performed by transforming shRNA plasmid along with lentivirus packaging plasmids in 293T cells, filtering (.45 micron) supernatant, and infecting a new population of 293T cells. After 48 hours, infected cells were Puromycin selected (1 ug / mL) for 10 days, after which RNA and protein were harvested.
|
| Growth protocol |
293T cells were grown in standard media (DMEM + 10% FBS + 1X Penicillin Streptomycin).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by standard Trizol extraction.
|
| Label |
biotin
|
| Label protocol |
Microarray sample preparation was performed using Ambion WT Expression Kit (Thermo Fisher Scientific). Labeling was performed using GeneChip Hybridization, Wash, and Stain Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
Hybridization and washing was performed as described in the GeneChip WT PLUS Reagent Kit (Affymetrix).
|
| Scan protocol |
Scanning was performed on a 7G scanner and imaged with the AGCC portal.
|
| Data processing |
Cel files were analyzed using Expression Console (build 1.4.1.46, Affymetrix), with RMA normalization and DABG probe-level detection. Only probesets with detection p-value ≤ 0.05 in more than half of the microarray samples were considered for downstream analysis. All probes corresponding to cassette exons profiled on the microarray (comprising exclusion junction, upstream and downstream inclusion junction, and inclusion exonic probes) were identified and normalized against the average signal on a per-gene basis to remove gene expression changes. Student’s t-test was performed on residuals for inclusion probes and exclusion probes separately to identify robust splicing changes, which were quantified by SepScore ( defined as the normalized change in exclusion minus the normalized change in inclusion).
HTA-2_0.r1.pgf
HTA-2_0.r1.Psrs.mps
RMA probeset-level signal estimates, DABG detection flag (Absent or Present), and DABG detection p-value obtained from 'Alt Splice Analysis' performed in Affymetrix Expression Console build 1.4.1.46.
|
| |
|
| Submission date |
Jan 28, 2016 |
| Last update date |
Mar 28, 2016 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL17585 |
| Series (2) |
| GSE77339 |
Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites [array] |
| GSE77634 |
Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites |
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