GSM5570848: h1;met1+/+ segregated from h1;met1+/- plant #2 (bisulfite); Arabidopsis thaliana; Bisulfite-Seq
gDNA for bisulfite sequencing was extracted with Dneasy plant mini kit according to manufacturer's protocol. 500 ng total gDNA was sheared to 100-1000bp DNA using Bioruptor Pico (Diagenode), then purified with 1.2X volume of SPRI beads (Beckman Coulter, cat. No. A63881). Fragmented gDNA was ligated to NEBNext Adaptor for Illumina using NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, ca. No. E7645). We performed bisulfite conversion twice with ligated libraries (QIAGEN, cat. No. 59104) to prevent imperfect conversion (<99% conversion) of unmethylated cytosines. Converted libraries were subjected to SPRI beads purification with 0.8X volume of beads. We amplified bisulfite-converted libraries with NEB next indexing primers (New England Biolabs Inc. cat. No. E7335S).