title: 4f-SAMMY-seq (Sequential Analysis of MacroMolecules Accessibility) and total RNA-seq data from prostate biopsies of 25 chemo-naïve patients (7 healthy biopsies and 18 tumor biopsies)
design: 4f-SAMMY-seq (Sequential Analysis of MacroMolecules Accessibility) and total RNA-seq data from prostate biopsies of 25 chemo-naïve patients (7 healthy biopsies and 18 tumor biopsies)
library_construction_protocol: Our cohort includes chemo-naïve patients followed in the Urology Division of Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico (Milan) who underwent the transrectal ultrasound-guided systematic sampling of prostate tissue (TRUS biopsy). This diagnostic procedure was performed as part of routine clinical management following the detection of abnormal digital rectal examination or an elevated PSA blood level. According to the current standard for the detection of PCa, 14 ultrasound-guided biopsy cores (diagnostic biopsies), seven from each side of the prostate gland, were collected from each patient for the clinical diagnosis. During the same procedure, one additional biopsy core to be used for our research project (research biopsy), was taken from a site directly adjacent to one of the diagnostic biopsies. Samples selected for epigenome studies consist of 17 fresh biopsies divided on the bases of the histology and the spatial distribution of the positive cores in two different groups: 10 PCa biopsies with histologically confirmed prostate cancer and 7 who had no cancer in any biopsy core.  Chromatin fractions (S2, S3 and S4) were diluted 1:2 in 1X TE buffer (10mM TrisHCl pH 8.0, 1 mM EDTA) and incubated with 3 U of RNAse cocktail (Ambion, AM2286) at 37° for 90 minutes, followed by 40μg of Proteinase K (Invitrogen, AM2548) at 55° for 150 minutes. Genomic DNA was then isolated using phenol/chloroform (Sigma-Aldrich, 77617) extraction, followed by a back extraction of phenol/chloroform with additional volume of TE1X. DNA was precipitated in 2 volumes of cold ethanol, 0.3M sodium acetate and 20ug glycogen (Ambion AM9510) for 1 hour on dry ice or overnight at -20°C. Dry pellets were resuspended in 50 ul (S2) or 15 ul (S3 and S4) of nuclease-free water and incubated at 4°C overnight. On the next day, S2 was further purified using PCR DNA Purification Kit (Qiagen, 28106) and separated using AMPure XP paramagnetic beads (Beckman Coulter, A63880) with the ratio 0.90/0.95 to obtain smaller fragments conserved as S2S (< 300 bp) and larger fragments labelled as S2L (> 300bp) fractions. Both were resuspended in 20 ul of nuclease-free water and then reduced to 15ul using a centrifugal vacuum concentrator. S2L, S3 and S4 fractions were sonicated in a Covaris M220 focused-ultrasonicator using screw cap microTUBEs (Covaris, 004078) to obtain a smear of DNA fragments peaking at 150-200 bp (waterbath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50, 150 seconds for S2L and 175 seconds for S3 and S4). Fractions were quantified using Qubit 4 fluorometer with Qubit dsDNA HS Assay Kit (Invitrogen, Q32854). Libraries were generated from each sample using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and Unique Dual Index NEBNextMultiplex Oligos for Illumina (NEB, E6440S). Libraries were then qualitatively and quantitatively checked on and run on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent, 5067-4626).