|
| Status |
Public on Mar 26, 2019 |
| Title |
Kelly_100THZ_2 |
| Sample type |
RNA |
| |
|
| Source name |
Kelly cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Kelly
treatment: THZ531 100nM
|
| Treatment protocol |
Cells were incubated for 6 h in the presence of DMSO or THZ531 (100nM, 400nM) before performing RNA extraction.
|
| Growth protocol |
IMR-32 and Kelly cells were grown in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Biological duplicates (equivalent to 5 x 10e6 cells per replicate) were collected and homogenized in 1ml of TRIzol Reagent (Ambion), purified using the mirVANA miRNA isolation kit (Ambion) following the manufacturer’s instructions and re-suspended in 50 ml nuclease-free water (Ambion). Total RNA was spiked-in with ERCC RNA Spike-In Mix (Ambion), treated with DNA-freeTM DNase I (Ambion) and analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies) for integrity.
|
| Label |
biotin
|
| Label protocol |
Biotinylated aRNA was prepared according to the standard Affymetrix protocol from 100 ng of total RNA (GeneChip 3' IVT Express Kit User Manual, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 12.5 ug of aRNA were hybridized at 45C for 16 hr at 60RPM on GeneChip Arrays (PrimeView). GeneChips were washed and stained in the Affymetrix Fluidics Station 450 according to the manurfacturer's instructions, using the buffers provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit.
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000 and images were extracted with Affymetrix GeneChip Expression Console.
|
| Data processing |
Microarray data were analyzed using a custom CDF file (GPL16043) that contained the mapping information of the ERCC probes used in the spike-in RNAs. The data were analyzed with Bioconductor using the MAS5 normalization method. Briefly, all chip data were imported in R (version 3.1.3) using the affy package, converted into expression values using the expresso command, normalized to take into account the different numbers of cells and spike-ins used in the different experiments and renormalized using loess regression fitted to the spike-in probes.
|
| |
|
| Submission date |
Apr 18, 2018 |
| Last update date |
Mar 26, 2019 |
| Contact name |
Ruben Dries |
| E-mail(s) |
[email protected]
|
| Organization name |
DFCI
|
| Department |
Computational Biology
|
| Lab |
Longwood Center
|
| Street address |
360 Longwood Ave
|
| City |
Boston |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL16043 |
| Series (2) |
| GSE113311 |
Global gene expression analysis in DMSO and THZ531 treated IMR32 and Kelly cells |
| GSE113314 |
Gene expression profiling in neuroblastoma cell lines treated with THZ531 or DMSO |
|
