GSM7848932: Control_M6; Homo sapiens; Bisulfite-Seq
Oragene OC-175 ORAcollect for pediatric samples and Oragene DNA Purification kit (Genotek, OGR-500) for adult samples Reduced Representation Bisulfite Sequencing libraries were generated as described in Gu et al. 2011. Bruefly, between 100-200 ng of genomic DNA was digested for 2 hours with MspI restriction enzyme (New England Biolabs) which cuts the DNA at every occurrence of the CCGG sequence, resulting in fragments starting with a CpG site. Libraries were prepared with the NEBNext Ultra II Modules (New England Biolabs) and the NEBNext Methylated Adaptor (New England Biolabs). The ligated DNA was size-selected using Ampure XP magnetic beads (Beckman Coulter) to produce a final library size of approximately 200 bp. Bisulfite conversion was performed with the EZ DNA Methylation-Gold Kit for single use (D5005, Zymo Research), followed by PCR amplification with the NEBNext Q5U polymerase and NEBNext Multiplex Oligos for Illumina (New England Biolabs) to barcode each library.