ChIP-qPCR Protocol for Chromatin Profiling

ChIP was performed following the instruction from a previously published study with minor modifications^{52 (link)}. In brief, 1 g of 10-day-old seedlings were cross-linked with 1% formaldehyde for 20 min under vacuum and ground into fine powder in liquid nitrogen. Chromatin was isolated and sheared into 200- to 500-bp fragments by sonication. The sonicated chromatin was incubated with 2 μg of anti-H3K9me2 (Abcam, ab1220), anti-H3 (Abcam, ab1791), or anti-Flag (Sigma, F1804) antibody and with 25 μl of Dynabeads Protein G (Invitrogen, 10003D) for 5 h with rotation at 4 °C. The precipitated chromatin DNA was then recovered and purified with a standard phenol–chloroform method. ChIP-qPCR was performed and results were calculated as percentage of input DNA. Sequences for the primers used for ChIP-qPCR are listed in Supplementary Data 7.