Reduced Representation Bisulfite Sequencing

EpiQuest libraries were prepared from 200–500 ng mouse genomic DNA obtained from primary cells (Sca1⁺Lin⁻ cells from bone marrow, and B220⁺ cells and Gr1⁺Mac1⁺ from peripheral blood) purified from Sca1-Bcl6^Δ mice and/or wild-type mice. DNA were pooled from 6–9 mice to provide one pooled replicate per condition, as performed previously^{65 (link),66 (link)}.The DNA was digested with 60 units of TaqI and 30 units of MspI (New England Biolabs) sequentially. Size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled in and 3'-terminal-A extended, extracted with a DNA Clean & Concentrator kit (Zymo Research). Ligation to pre-annealed adapters containing 5'-methylcytosine instead of cytosine was performed using the Illumina DNA preparation kit and protocol. Purified, adaptor ligated fragments were bisulphite-treated using the EZ DNA Methylation-Direct Kit (Zymo Research). Preparative-scale PCR (18 cycles) was performed and purified PCR products were subjected to a final size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size were excised. Library material was recovered from the gel using a Zymoclean Gel DNA Recovery Kit (Zymo Research) and sequenced on an GAIIx genome analyzer (Illumina), yielding between 43,930,708 and 60,139,994 total reads for each condition.