title: GSM5928715: h1ddm1.f4.bsseq; Arabidopsis thaliana; Bisulfite-Seq
library_construction_protocol: DNA was isolated by pulverizing ~0.5 g of flash frozen rosette leaves of 4 week post-germination plants.  ~100 mg of resulting powder was used to extract genomic DNA (gDNA) with the DNeasy plant mini kit (Qiagen cat. no. 69104) per manufacturer's instructions. gDNA was subsequently sonicated with the Bioruptor Pico (Diagenode) to ~250 bp median fragment length using 10 cycles of 30 seconds on and off.  Agencourt Ampure beads (referred to as “beads” henceforth, cat. no. A63881) were then used at 2X volume to purify the sheared DNA. Following ligation of methylated Truseq sequencing adapters (Illumina) to sheared DNA, bisulfite conversion of DNA was carried out according to manufacturer's protocol (Qiagen Epitect Kit, cat. no. 59104) except without using carrier RNA.  DNA was purified twice with 1.2X beads and converted a second time to ensure complete bisulfite conversion of unmethylated cytosine. Libraries were constructed using NEBnext kits (NEB cat. no. E7645) or Nugen/Tecan Ovation Ultralow (cat. no. 0344NB-08) following the manufacturer's instructions.   NEB next indexing primers (cat. no. E7335S) were used for generating multiplexed libraries during PCR amplification of libraries.