Study of vitamin D signaling pathways during the initiation of prostate cancer
To determine the mechanism underlying the role of VDR in prostatic epitheliales cells during prostate cancer, bulk RNA- sequencing will be performed on sorted luminal cells from PTEN(i)pe-/- and PTEN/VDR(i)pe-/- mouse 1M-3M AGI. Overall design: Total RNA was isolated from FACS-isolated luminal cells using TRI Reagent (Molecular Research Center, Inc.) and RNeasy Kits (Qiagen). cDNA libraries were generated from 600 ng RNA with an integrity number > 8 using the Stranded mRNA-seq Lib Prep Kit (Illumina), according to the manufacturer's instructions, quantified and checked for quality using capillary electrophoresis. Fifty base pair single-read sequencing was performed on a NextSeq 2000 (Illumina) following the manufacturer's instructions. Reads preprocessing steps were performed using cutadapt 1.10, reads were mapped onto the mm10 assembly of Mus musculus genome using STAR version 2.5.3a. Gene expression quantification was performed from uniquely aligned reads using htseq-count version 0.6.1p1, with annotations from Ensembl version 108 and “union” mode. Only non-ambiguously assigned reads were retained for further analyses.