Ht 12 v4 beadchip array

RNA was extracted from CWR22Rv1 cells grown in steroid-depleted media subjected to control, AR or FOXA1 knockdown for 24 hours prior to vehicle or 1 μM Enzalutamide treatment for 24 hours. Samples were hybridised onto an Illumina HT12 v4 BeadChip Array (performed by The Wellcome Trust Centre for Human Genetics, Oxford University. Array processing, normalisation and quality control checks were performed using the R package ‘Lumi’. Probes intensity values were converted to VSD (variance stabilised data) using variance stabilizing transformation. The robust spline normalisation (RSN) was used as the array normalisation method. Outlier samples, poor quality probes (detection threshold < 0.01), and probes that are not detected at all in the remaining arrays were removed prior downstream analysis. The remaining probe (22,551) normalised intensity, VSD was used in the differential expression analysis. Differential expression analysis was performed using the R package ‘Limma’, and P values were adjusted to control the false discovery rate (FDR) using the Benjamini–Hochberg method. Gene ontology analysis was performed as described in [45 (link)].

Gene expression levels for FKBP5 at all time‐points were taken from previously quality‐control processed data from whole blood samples quantified using the Illumina HT‐12 v4 BeadChip array (n = 102). Full details of data preparation and quality‐control procedures are reported elsewhere (Roberts et al., 2017). Briefly, this involved background correction and filtering based on probe intensity and detection rates. Probes were then transformed and normalized, and outliers identified and removed.

RNA was prepared using the RNeasy mini kit (Qiagen) per the manufacturers instructions. cRNA was amplified using the Illumina TotalPrep RNA Amplification Kit (Ambion) and run on an Illumina HT_12_v4 BeadChip Array (Ilumina), as per the manufacturer’s instructions. Analysis of microarray data was performed using Genome Studio software (Illumina).

Genomic DNA was prepared using the DNA Mini Kit, and RNA with the RNeasy Mini Kit, as per the manufacturer's instructions (Qiagen). qPCR was carried out on a Stratagene MX3000P QPCR machine (Agilent technologies) utilizing 40 cycles. cRNA for was amplified using the Illumina TotalPrep RNA Amplification Kit (Ambion) and ran on an Illumina HT_12_v4 BeadChip Array (Ilumina), as per the manufacturer's instructions. Oligos are found in Fig S6.

For each replicate, probes were synthesized using the Illumina TotalPrep-96 RNA Amplification Kit and hybridized to Illumina HT-12 v4 BeadChip Arrays using standard Illumina procedures (Cambridge Genomic Services, University of Cambridge, U.K.). Three replicates were performed for each W12 clone sample, to control for variation in labeling and hybridization efficiency. The raw intensities files are available at ArrayExpress (Accession number: E-MTAB-4409).