Fine mapping of androgen regulated genes in LNCaP cells

Detailed analysis of androgen regulated gene expression in the LNCaP prostate cancer cell line. Since androgens and the AR are known to be important for prostate cancer cell proliferation and invasion we aimed to identify androgen receptor (AR) regulated genes by combining this detailed Illumina beadarray study of androgen regulated gene expression with AR ChIP-sequencing data. LNCaP cells were grown in RPMI medium supplemented with 10% charcoal dextran stripped (steroid depleted) FBS for 72h, before treatment with 1nM R1881 or vehicle control. Total RNA was harvested every 30min for 4h and then every hour up to 24h using trizol (Sigma), quantified using a Nanodrop spectrophotometer (ND-1000). RNA samples were prepared for analysis on Illumina Human 6 v2 BeadArrays and Biotrove Realtime PCR panels according to the manufacturers protocols (see Supplementary Methods for details). Expression array data were normalised in R using the beadarray software and BASH (see Supplementary Methods for details) (Cairns et al., 2008; Dunning et al., 2007). 48 total RNA samples from LNCaP cells grown for 72h in RPMI supplemented with 10% charcoal dextran stripped FBS, comprising: 3 time zero samples; 10 vehicle (ethanol) control samples taken at 2h, 4h, 8h, 12h, 24h in duplicate; 36 androgen (R1881) treated samples taken every 30min for 4h then every hour until 24h following treatment (with replicates at 1h, 2h, 4h, 8h, 12h, 16h, 20h, 24h) The Illumina HumanWG v2 BeadArrays consist of two replicate sections that we treat as technical replicate arrays for the purposes of this analysis due to small but systematic shifts between sections that need to be addressed in the normalization. Data were analysed from the raw bead-level using the beadarray software, with spatial artefacts identified and removed automatically (BASH) and curated manually (Cairns et al., 2008; Dunning et al., 2007)