RRBS of ALCL patient
For reduced representation bisulfite sequencing (RRBS), 100 ng of genomic DNA were digested for 6h at 65C with 20 U TaqI (New England Biolabs, R0149L) and 6h hours at 37C with 20 units of MspI (New England Biolabs, R0106L) in 30 l of 1x 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- (New England Biolabs, M0212L) and dNTP mix (10 mM dATP, 1 mM dCTP, 1 mM dGTP). After ligation to methylated Illumina TruSeq LT v2 adaptors using T4 DNA Ligase rapid (Enzymatics, L6030-HC-L)), the libraries were size selected by performing a 0.75x clean-up with AMPure XP beads (Beckman Coulter, A63881). The libraries were pooled based on qPCR data and subjected to bisulfite conversion using the EZ DNA Methylation Direct Kit (Zymo Research, D5020) with changes to the manufacturers protocol: conversion reagent was used at 0.9x concentration, incubation performed for 20 cycles of 1 min at 95C, 10 min at 60C and the desulphonation 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 KAPA HiFi HS Uracil+ RM (Roche, 7959079001). The minimum number of enrichment cycles was estimated based on a qPCR experiment. After a 1x AMPure XP clean-up, quality control was performed by a Qubit dsDNA HS (Life Technologies, Q32854) and Agilent High Sensitivity DNA Kit (reorder number) (Agilent, 5067-4626).