Bisulfite-Seq of soybean: germinated cotyledons
Cotyledons of soybean WT and cco mutant (six-days-old after germination) were collected for genomic DNA extraction using a DNAsecure Plant Kit (TianGen, Beijing, China). The quality of DNA samples was evaluated with agarose gel and Nanodrop ND1000 spectrophotometer (Nanodrop, Wilmington, DE). The concentration of DNA samples was measured with Qubit 2.0 fluorometer (Life Technologies, CA, USA). Then, DNA samples were randomly fragmented to 100-300bp with a sonicator (Covaris S220, Thermo Scientific, USA). After end repair and 3-terminal-A extension, cytosine-methylated sequencing adaptors were ligated to DNA fragments according to Illumina manufacturers instructions. Further, bisulfite conversion was conducted using EZ DNA Methylation-Gold Kit (Zymo Research, CA, USA), and DNA library was carried out by PCR amplification. The concentration and size of DNA library were confirmed by Qubit2.0 fluorometer (Life Technologies, CA, USA) and biological analyzer Agilent 2100 (Agilent, CA, USA), respectively. Finally, pair-end sequencing was performed using Illumina Hiseq2500 platform.