Affymetrix genechip 3000

Total RNA was isolated from cells using an RNeasy Plus Mini Kit (74136, Qiagen). RNA quality was analysed with an Agilent 2100 Bioanalyzer and an RNA 6000 Nano Kit (5067-1511, Agilent). RNA samples showing RNA integrity numbers ≥7 were used to generate biotinylated complementary RNA (cRNA) with a GeneChIP WT Plus Reagent Kit (902280, Affymetrix) and GeneChip Hybridization, Wash and Stain Kit (900720, Affymetrix). Labelled cRNA was hybridized to Affymetrix Mouse Gene 1.0 ST microarrays (Affymetrix), then stained and scanned with an Affymetrix GeneChip 3000 (Affymetrix).

Ovcar8 cells were transfected with either siNeg or siBRD4 in 3 independent plates and harvested 48hr post-transfection. For chemical inhibition, JQ1 was added for 24hr at final concentration 0.5μM in Ovcar8; DMSO was used for control in 3 independent plates. Total RNA was isolated using RNeasy with on-column DNase treatment (Qiagen). Microarray analysis was previously described [2 (link), 6 (link)]. Briefly, samples were prepared, labeled and hybridized to Affymetrix H133Plus2.0 gene chips and scanned on Affymetrix GeneChip 3000 (Affymetrix, Santa Clara, CA). CEL files were imported into GeneSpring (Agilent); probe levels were normalized by GC-RMA algorithm, and entities with intensity values <100 were filtered out before performing significance analysis. Gene set enrichment analysis (GSEA) was performed in the drug treated group and the siRNA group. Hallmark signatures shared in common between these two groups with false discovery rate (FDR) <0.1 [31 (link)].