|
| Status |
Public on Mar 18, 2018 |
| Title |
FUS-silenced SK-N-BE cells, biological rep 2 (transcription-level) |
| Sample type |
RNA |
| |
|
| Source name |
siFUS
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: neuroblastoma SK-N-BE
treatment: 96-hour FUS gene silencing
|
| Treatment protocol |
For gene silencing of TDP-43 and FUS RBPs, SK-N-BE cells were transfected for 96 hours with 80nM of specific siRNA duplexes (Darmakon and Invitrogen, respectively) in a double round of transfection using Lipofectamine 2000 (Invitrogen). Stealth siRNA low GC (Invitrogen) was used as a negative control.
|
| Growth protocol |
The human neuroblastoma SK-N-BE cell line was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1mM sodium pyruvate, 2 mM l-glutamine, 4.5 g/l glucose and 100units/ml penicillin and 100g/ml streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Plus Mini kit (Qiagen) from TDP-43, FUS or control mock siRNA-treated SK-N-BE cells.
|
| Label |
biotin
|
| Label protocol |
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
|
| |
|
| Hybridization protocol |
Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99° for 5 minutes, then 42° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). • Transfer 200μl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 45° at 60rpm• Fluidics washing is FS450_0001
|
| Scan protocol |
Affymetrix Gene ChIP Scanner 3000 7G
|
| Description |
FUS_4
|
| Data processing |
Data were processed using EASANA from GenoSplice technology. GC background correction were applied to probe intensities (core), gene and exon level expression were summarized from the corrected intensities and then quantile normalized.
HuEx-1_0-st-v2.r2.pgf
HuEx-1_0-st-v2.r2.dt1.hg18.core.mps
|
| |
|
| Submission date |
Mar 18, 2015 |
| Last update date |
Mar 18, 2018 |
| Contact name |
Pierre de la Grange |
| E-mail(s) |
[email protected]
|
| Organization name |
GenoSplice technology
|
| Department |
Paris Biotech Santé
|
| Street address |
29, rue du Faubourg Saint-Jacques
|
| City |
Paris |
| ZIP/Postal code |
75014 |
| Country |
France |
| |
|
| Platform ID |
GPL5175 |
| Series (1) |
| GSE67027 |
Splicing events and transcriptome changes upon TDP-43 and FUS knock-down in human neuroblastoma cells |
|
| Relations |
| Alternative to |
GSM1636399 (transcript-level analysis) |
