title: Illumina HiSeq 2500 sequencing; DNMT3A epigenetically regulates key microRNAs involved in epithelial-to-mesenchymal transition in prostate cancer
design: DNMT3A epigenetically regulates key microRNAs involved in epithelial-to-mesenchymal transition in prostate cancer
library_construction_protocol: Human PCa cell lines, PC3 (RRID:CVCL_0035)  were obtained from American type culture collection (ATCC, Manassas, VA, USA) and regularly tested for mycoplasma (N-GARDE Mycoplasma PCR Reagent set, EuroClone,  Milan, IT). Both cell lines were cultured in RPMI 1640 (EuroClone) supplemented with 10% heat-inactivated foetal bovine serum (EuroClone), 2 mM glutammine and 100 U/ml penicillin/ streptomycin (EuroClone). Total RNA was isolated from cells with TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). For mRNA quantification, complementary DNAs were prepared retro-transcribing 1 μg of total RNA with iScript Reverse Transcription supermix for reverse transcriptase–quantitative PCR (Bio-Rad, Hercules, CA, USA). Quantitative reverse transcriptase–PCR reactions were performed with iQ SYBR Green supermix (Bio-Rad). The PCR was monitored in real-time using a MJ-Chromo4 (Bio-Rad) that allowed determination of the threshold cycle (Ct) at the beginning of the exponential amplification of the PCR products. The average Ct, from triplicate assays, was used for further calculations. For small RNA sequencing, 1 ug of total RNA was used for indexed sequencing library preparation with a TruSeq small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced on HiSeq1500 (Illumina) for 50 cycles