Bisulfite-treated DNA library construction and sequencing

Genomic DNA was extracted from cotton ovules and fibres using the CTAB method (30). For each sample, a total of 2 μg DNA was fragmented to 200–500 bp by sonication. DNA libraries were constructed using the Illumina TruSeq DNA Sample Prep Kit following the manufacturer's instructions with minor modifications necessary for the bisulfite treatment. Briefly, the fragmented DNA was processed by repairing ends, adenylating 3′ ends and ligating adapters. Adapter-ligated DNA fragments were then treated with bisulfite, followed by 10 cycles of PCR amplification. Finally, the bisulfite-treated PCR products were purified using AMPure XP Beads. Bisulfite conversion of DNA samples was conducted using the EZ DNA Methylation-Gold™ Kit (Catalog No. D5005). The unmethylated lambda DNA (Promega) was converted simultaneously as a control. The paired-end sequencing of bisulfite-treated DNA libraries was performed using the Illumina HiSeq 2000 system (paired-end 100-bp reads).