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Sample GSM980420 Query DataSets for GSM980420
Status Public on Sep 10, 2012
Title TDP43OE-Flp-In-293 hjay 3
Sample type RNA
 
Source name human Flp-In-293 cells
Organism Homo sapiens
Characteristics cell line: HEK293
treatment: TDP43 Over Expression
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

Data table header descriptions
ID_REF
VALUE Quantile normalized and gc background corrected.

Data table
ID_REF VALUE
58017 2060.92611
28336 412.81782
564321 51.88564
544357 2392.35958
342596 183.64522
81834 443.15584
67525 1.8137
474416 4741.14217
403345 156.04535
440294 183.08212
296021 186.57094
150275 1642.73666
564868 2689.34808
287374 1137.59691
198359 64.58496
399155 60.01786
557453 158.74449
220912 204.77904
313072 1566.88572
19092 1720.79921

Total number of rows: 569393

Table truncated, full table size 9243 Kbytes.




Supplementary file Size Download File type/resource
GSM980420_TDP43OE-Flp-In-293_hjay_3.CEL.gz 27.2 Mb (ftp)(http) CEL
Processed data included within Sample table

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