|Public on Jun 30, 2016
|colorectal carcinoma cell line
|cell line: vorinostat-resistant HCT116
treatment: siPSDM13 DMSO
|Cells were reverse transfected in 12 well plates with the appropriate siRNA (final concentration 40nM, Dharmacon siGENOME SMARTpools, Thermo Fisher) or lipid only as a control (mock transfection). Each well contained 3.5 x 10^5 cells and 1.0ul of Dharmafect 2 (Thermo Fisher). Media was refreshed after 24hrs and after 48hrs, cells were treated with 2.5uM vorinostat or DMSO (solvent) as a control. Cells were harvested by trypsinisation after 4hrs, 8hrs or 12hrs of drug treatment.
|Cells were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% sodium pyruvate, 2.5g/L glucose.
|RNA was extracted from cells using the Rneasy RNA extraction kit (QIAGEN) as per the manufacturer's protocol. RNA was quantitated using spectrophotometry (Nanodrop).
2ug RNA was prepared for sequencing using the TruSeq RNA Sample Preparation v2 Guide with all kit reagents purchased from Illumina.
|Illumina HiSeq 2500
|Illumina Casava 1.8.2 software used for basecalling.
Reads were quality checked by FastQC and trimmed if necessary for low base quality or adaptor, and then mapped to the human reference genome (GRCh37) using Tophat2 v.2.0.8b with maximum number of multiple hits set to 1 and using the option to map first to the reference transcriptome (Ensembl v69).
Counts per gene were obtained using HTSeq and normalised using the limma-voom method.
Supplementary_files_format_and_content: tab-delimited text file containing limma-voom normalised log2 values for each Sample
|May 21, 2014
|Last update date
|May 15, 2019
|Katrina Joy Falkenberg
|Peter MacCallum Cancer Centre
|Gene Regulation Laboratory
|St Andrews Place
|RNA-seq analysis of vorinostat-resistant HCT116 cells following gene knockdown of GLI1 or PSMD13 with or without vorinostat treatment