|
| Status |
Public on Sep 10, 2012 |
| Title |
LIN28V5-293 hjay 2 |
| Sample type |
RNA |
| |
|
| Source name |
human LIN28-V5 293 stable cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
treatment: LIN28 Flp-In-293
|
| Treatment protocol |
To generate the LIN28-V5 293 stable cell line, pEF5/FRT/LIN28-V5 plasmid was co-transfected along with the FLP Recombinase expressing plasmid pOG44 into Flp-In-293 cells using Lipofectamine-2000 (Life Technologies) according to the manufacturer’s instructions. Recombination and insertion of LIN28-V5 at the Flp-In site confers stable integration and expression of the LIN28-V5 fusion protein and hygromycin resistance. Stably transected clones were selected and propagated in media supplemented with 75-100 μg/ml hygromycin B (Life Technologies). To achieve knockdown of LIN28 in hES cells, we utilized an shRNA construct targeting human LIN28A in the pLKO.1 vector (TRCN0000102579; Open Biosystems). As a control, a pLKO.1 vector containing an shRNA toward GFP was used (Open Biosystems). Lentivirus expressing these constructs was prepared in 293T cells, as previously described (Yeo et al., 2009). Human ES cells were treated with Accutase to generate single cell suspensions and infected with LIN28 or matched control GFP virus. The media was changed daily and cells were harvested 72 hours after infection. To acheive TDP-43 over expression, Flp-In-293 cells were grown to ~70% confluency in 6-well cell culture plates and transfected with 4μg of TDP-43-GFP (Liu-Yesucevitz et al., 2010) or control pEGFP-C2 (AddGene) plasmid using Lipofectamine-2000 (Life Technologies) according to manufacturer’s instructions. After 48 hours cells were harvested
|
| Growth protocol |
HEK293 cells containing an integrated Flp-In site (Flp-In-293; Life Technologies) were cultured in DMEM media supplemented with 10% FBS and 2mM L-glutamine and passaged using TrypLE (LifeTechnologies). Human ES cell-lines H9 and HUES6 were grown in feeder-free conditions with mTeSR media (Stemcell Technologies) and on Matrigel (BD Biosciences) for support. Cells were passaged manually or with Dispase (BD Biosciences) every 5-7 days.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Trizol (Life Technologies) extraction of RNA was performed according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
WT Terminal Labeling Kit, Affymetrix
|
| |
|
| Hybridization protocol |
Hybridization cocktails containing ~5.5 ug of fragmented and labeled DNA target were prepared and applied to GeneChip Human Splice Junction arrays. Hybridization was performed for 16 hours using the Fluidics 450 station.
|
| Scan protocol |
Arrays were scanned using the Affymetrix 3000 7G scanner and GeneChip Operating Software version 1.4 to produce .CEL intensity files.
|
| Data processing |
Data processed using Affymetrix package (Affy Power Tools) apt-probeset-summarize. Iter-plier algorithm used to quantify probesets.
HJAY_r2.pgf
|
| |
|
| Submission date |
Aug 02, 2012 |
| Last update date |
Sep 10, 2012 |
| 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 |
GPL15106 |
| Series (2) |
| GSE39855 |
LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance (splicing array) |
| GSE39873 |
LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance |
|
