title: WGBS (CRX1930299; CRA029290)
library_construction_protocol: Genomic DNA was extracted from young leaves of rye (Secale cereale L.) using a standard CTAB method. The WGBS library was constructed using the MGIEasy Bisulfite Sequencing Library Prep Kit (MGI) according to the manufacturer's instructions. Briefly, 200 ng of high-quality genomic DNA per sample was fragmented by ultrasonication (Covaris, USA) to an average size of 200-500 bp. The fragmented DNA was then end-repaired, adenylated at the 3' ends, and ligated with methylated adapters. Post-ligation purification was performed using MGIEasy DNA Clean Beads (MGI). The adapter-ligated DNA underwent bisulfite conversion using the EZ DNA Methylation-Lightning Kit (Zymo Research, USA), which deaminates unmethylated cytosines to uracils while leaving methylated cytosines unchanged. The bisulfite-converted DNA was then amplified by PCR using specific primers. The final library was purified and quality-checked for fragment size distribution using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and quantified by Qubit Fluorometer (Invitrogen, USA). Qualified libraries were sequenced on the DNBSEQ-T7 platform (MGI) to generate paired-end reads for subsequent methylation analysis.