ChIP-seq Analysis of H3K27me3 and H3K4me3

ChIP-seq of trifoliate leaves of W05 was performed following the procedures of a previous study with minor modifications [24 (link)]. ChIP-seq grade anti-H3K27me3 (Diagenode, C15410195, Denville, NJ, USA) and anti-H3K4me3 (Millipore, 07473, Burlington, VT, USA) antibodies were used for chromatin immunoprecipitation. About 5 ng ChIP’ed DNA or input DNA was used for ChIP-seq library construction via TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501, Nanjing, China). ChIP-seq libraries were sequenced in PE150 mode of the Illumina X Ten platform. Adaptor trimming, low-quality reads removing, and mapping were performed using TrimGalore and Bowtie2. The mapped reads (>MAPQ30) were used for peak-calling using MACS2 [25 (link)], and the parameters were set as ‘—trackline —extsize 147 —broad -q 0.01 —nomodel -g 1.0e + 9 —buffer-size 500,000’. An input library was used as control.