EG4 BS-Seq replicate 2: Comparative transcriptome analysis among closely related rice strains
Genomic DNA (1-3 μg) isolated from seedlings (DNeasy Plant Mini kit; Qiagen) was sheared  (with a Covaris instrument to mean size of 300 bp), fragments purified, end repaired, A-tailed, and ligated with the methylated adapters (BiooScientific) following instruction from KAPA LTP library preparation Kit (Kapa Biosystems). Bisulfite treated DNAs (Epitect Bisulfite kit; Qiagen) were amplified for 12-16 cycles and libraries were applied to paired-end sequencing with read length of 100nt on the Illumina Hiseq 2500 platform. Raw reads were quality trimmed and aligned to the NB reference genome (MSU7) (http://rice. plantbiology.msu.edu) using Bismark (Krueger and Andrews, 2011) allowing two mismatches. EG4 reads were also mapped to the mPing pseudogenome (mPing sites with 1kb flanking sequences). Bismark’s methylation extractor script was used to calculate the methylation level for each cytosine. The bisulfite conversion rate for each library was calculated based on the methylation level of cytosines from reads mapping to the unmethylated chloroplast genome.
University of California, Riverside
EG4 bisulfite sequencing (BS-seq) replicate 2