Ribozero gold library prep kit

Prostates were harvested from Akap12^−/−;Pb-Cre:Rb1^fl/fl and Pb-Cre:Pten^fl/fl:Rb1^fl/fl mice by microdissection, snap frozen in liquid nitrogen, and stored at −80 °C. RNA was isolated from frozen tissue using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA samples were interrogated by the RPCCC Genomics Shared Resource using TruSeq Stranded Total RNA with the RiboZero Gold library prep kit (Illumina, San Diego, CA) with 1 μg input, sequenced on an Illumina HiSeq2500. Alignment to the mouse genome (mm10 version) was performed by the RPCCC Bioinformatics Shared Resource using RefSeq [43 (link)] and the UCSC Genome Browser [44 (link)]. Quality control for the raw reads was performed using fastqc [45 (link)] and adapter trimming was done using atropos [46 (link)]. Spliced alignments of reads to the reference genome was done using TopHat2 [47 (link)], allowing a maximum of 1 mismatch/read, and quality control for this alignment was done using RSeQC software [48 (link)]. The differential expression report was generated using DESeq2 [49 (link)] and the expression levels were normalized using Fragments Per Kilobase of transcript per Million mapped reads (FPKM). FASTQ files for the RNA seq data were uploaded to the Gene Expression Omnibus (GEO) under accession numbers GSE90891 and GSE242387.

For RNA-seq, 100 ng of total RNA was converted into cDNA libraries using the Illumina TruSeq Stranded Total RNA, as previously described (Wehmas et al.^{20 (link)} for Study 1; Hester et al.^{9 (link)} for Study 2A; Webster et al.^{8 (link)}: Study 2B). Ribosomal RNA was removed from samples through sequence-specific rRNA depletion using biotinylated probes and strepavadin bead immobilization (Ribo-Zero Gold Library Prep Kit, #RS-122-2303, Illumina, San Diego, CA). Samples were fragmented by heating with divalent cations. FFPE samples were subjected to reduced fragmentation/heating times to enable consistent library size distributions with FR. This process was quantified by Agilent Bioanalyzer (DNA 1000 kit #5067-1504) and qPCR (KAPA Library Quant Kit #KK4824, KAPA Biosystems, Wilmington, MA) followed by library normalization and sequencing. Samples were labeled with a barcode, mixed together in a sequencing pool, and run at eight per sequencing lane. Sequencing for Study 1, Study 2A, and Study 2B was performed at Expression Analysis (EA) (EA Genomic Services, Q2 Solutions—a Quintiles Quest Joint Venture, Durham, NC) using the Illumina HiSeq platform with 2 × 50 bp-paired, 2 × 50 bp-paired, and 1 × 50 bp-single end reads, respectively. Samples for these studies were run in independent batches.