Feature extractor software

Genomic DNA was isolated from transduced cells using the DNeasy Purification Kit (QIAGEN; Tokyo, Japan) according to the manufacturer's protocol. The integrated shRNAs prepared from LNCaP genomic DNAs were amplified using primers (Decode RNAi-GIPZ, annotated genes screening library-negative selection kit from Thermo Scientific) specific for the barcodes for the library plasmid DNA [13] (link), [14] (link), [25] (link). The PCR products were gel-purified using the QIAquick PCR purification Kit (QIAGEN). Purified DNA fragments (1.5 µg) from LNCaP cells treated with vehicle or bicalutamide were labeled with cyanine-3 (Cy3) or cyanine-5 (Cy5) dye, respectively, using the Genome DNA Enzymatic Labeling Kit (Agilent; Santa Clara, CA, USA) and purified by the removal of unbound cyanine dyes with an Ultracell YM-30 Microcon centrifugal filter device (Millipore Japan; Tokyo, Japan). Microarray hybridization was performed using the Oligo cDGH/ChIP-on-ChIP Hybridization Kit (Agilent). Agilent Feature Extractor software was used to scan microarray images. The GEO accession number for the microarray data is GSE60382. A volcano plot was generated by clustering based on probes. Depleted (fold change <0.5; P<0.01) signals in the bicalutamide-treated LNCaP cells compared with the vehicle-treated cells were selected as bicalutamide response-related genes [23] (link), [25] (link), [26] (link).

Total RNA was isolated from lung by homogenization (10 % w/v) in Trizol (Invitrogen, Carlsbad, CA) followed by chloroform extraction and isopropanol precipitation as previously described [23 (link)] and RNA quality assessed using a BioAnalyzer (Agilent Technologies, CA). Gene expression profiling experiments using Agilent Mouse Whole Genome 44 K microarrays and data processing/analysis was performed as previously described [23 (link)]. Briefly, fluorescent probes were prepared using Agilent QuickAmp Labeling Kit according to the manufacturer’s instructions. Spot quantitation was performed using Agilent’s Feature Extractor software and all data entered into a custom-designed database, SLIMarray (http://slimarray.systemsbiology.net), and then uploaded into Genedata Analyst 8.0 (Genedata, Basel, Switzerland). Data normalization was performed in Genedata Analyst using central tendency followed by relative normalization using pooled RNA from mock infected mouse lung (n = 6) as reference. Transcripts differentially expressed (at least 2-fold, p value < 0.01) between infected and control animals were identified by standard t test using the Benjamini-Hochberg procedure to correct for false positive rate in multiple comparisons. Ingenuity Pathway Analysis and Entrez Gene (www.ncbi.nlm.nih.gov/sites) were used for mammalian gene ontology and pathway analysis.