Illumina HiSeq 2500 paired end sequencing; DNA methylation variations are required for reversible EMT induced by cancer-associated fibroblasts in PCa cells
DNA methylation variations are required for reversible EMT induced by cancer-associated fibroblasts in PCa cells
Human prostate cancers cell lines, PC3  were obtained from American type culture collection (ATCC - Manassas, VA, USA) and authenticated by PCR/short tandem repeat (STR) analysis PC3 cells were cultured in DMEM medium containing 10% foetal bovine serum (FBS, EuroClone®), 2mM Glutammine and 100 U/ml of penicillin/streptomycin (EuroClone®). Human prostate fibroblasts (HPFs and CAFs) were isolated from surgical explantation after patient informed consent in accord with the Ethics Committee of the Azienda Ospedaliera Universitaria Careggi. Briefly, HPFs and CAFs were extracted from prostate of benign prostate hyperplasia-bearing patients and from intra-tumoural regions of prostate of PCa-bearing patients respectively. Tissues were digested overnight in 1 mg/ml collagenase I, and cells were plated in DMEM containing 10% FBS. PC3 induction were performed with a treatment for 72h with conditioned mediums (CM-HPF or CM-CAF) (CM from HPFs and CAFs was obtained by 48-h serum-starved cells, clarified by centrifugation, and freshly used) PC3 induction were performed with a treatment for 72h with conditioned mediums (CM-HPF or CM-CAF) (CM from HPFs and CAFs was obtained by 48-h serum-starved cells, clarified by centrifugation, and freshly used) Indexed libraries were prepared from 1 ug purified RNA with TruSeq Stranded Total RNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions