DNA was extracted from cells by DNA lysis buffer (100mM NaCl, 10mM Tris pH 8, 25mM EDTA,  0.5% SDS, 0.1mg/ml proteinase K).  DNA extracts were incubated overnight at 56oC.  DNA was purified by   2 rounds of phenol/chloroform  and precipitated with 2 volumes of ethanol and 1/10 volume of 3M sodium acetate solution. Libraries were prepared by the Zymo Epiquest service. RRBS libraries were prepared from 200-500 ng genomic DNA digested with 60 units of TaqI and 30 units of MspI (NEB) sequentially. Size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled in and 3’-terminal-A extended, extracted with Zymo Research DNA Clean & Concentrator™ kit. Ligation to pre-annealed adapters containing 5’- methyl-cytosine instead of cytosine (Illumina) 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 was performed and DNA Clean & Concentrator™ -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 (Zymoclean™ Gel DNA Recovery Kit) and sequenced on the Illumina HiSeq platform.
GEO Accession
GSM1447379