GSM9128185: Patient 07, Primary prostate cancer, PSMA low tumor area; Homo sapiens; Bisulfite-Seq
DNA was extracted automated with the Qiagen EZ1 using the Qiagen EZ1 DNA FFPE Kit following manufacturers instruction for sample preparation. Reduced Representation Bisulphite Sequencing (RRBS) libraries were generated as described previously. Briefly, up to 100 ng of genomic DNA were digested for 12 hours at 37 °C with 20 U of MspI (R0106L, New England Biolabs, Inc., Ipswich, MA, USA) in 30 μl of 1 x NEB buffer 2. To retain even the smallest fragments and to minimize the loss of material, end preparation and adaptor ligation were performed in a single-tube setup. End fill-in and A-tailing were performed by addition of Klenow Fragment 3' > 5' exo-mix (P7010-LC-L, Enzymatics) and dNTP mix (10 mM dATP, 1 mM dCTP, 1 mM dGTP, N0446S, New England Biolabs). After ligation to methylated Illumina TruSeq LT v2 adaptors using T4 DNA Ligase rapid (L6030-HC-L, Enzymatics), the libraries were size selected via a 0.75 x clean-up with AMPure XP beads (A63881, Beckman Coulter). The libraries were pooled based on qPCR data and subjected to bisulfite conversion using the EZ DNA Methylation Direct Kit (D5020, Zymo Research) with changes to the manufacturer's protocol: conversion reagent was used at 0.9 x concentration, incubation performed for 20 cycles of 1 min at 95 °C, 10 min at 60 °C and the desulfonation time was extended to 30 min. These changes increase the number of CpG dinucleotides covered, by reducing double strand break formation in larger library fragments. Bisulfite-converted libraries were enriched via KAPA HiFi HS Uracil+ RM (07959079001, Roche Molecular Systems, Inc.). The minimum number of enrichment cycles was estimated based on a qPCR experiment. After a 1 x AMPure XP clean-up, library concentrations were quantified with the Qubit Fluorometric Quantitation system (Q32854, Thermo Fisher Scientific, Waltham, MA, USA) and the size distribution was assessed using the High Sensitivity DNA Kit (5067-4626, Agilent, Santa Clara, CA, USA) on a 2100 Bioanalyzer (Agilent) instrument. Sample-specific RRBS libraries were diluted and pooled in equimolar amounts.