Chromatin Isolation and MNase Digestion

2 g of above ground tissue was harvested without crosslinking and nuclei and chromatin were isolated as previously described (Chodavarapu et al., 2010 (link)) with minor changes. The nuclear pellet was washed twice with HBB buffer. The isolated chromatin was digested with a final concentration of 0.2–0.5 units/μl MNase (Takara, Tokyo, Japan) for 3 min in digestion buffer at 37°C. Subsequent steps were performed as previously described (Chodavarapu et al., 2010 (link)). Relative nucleosome occupancy was analyzed by tiled oligo qPCR. Percent input enrichment for each primer pair was extrapolated using a dilution series of undigested genomic DNA (Gévry et al., 2009 (link)). Fold enrichment of nucleosome bound DNA was calculated by normalizing percent input of each primer pair over that of the gypsy-like retrotransposon (At4g07700). MNase in protoplasts was performed as in intact tissues with some modification. 2 × 10⁶ cells were harvest by centrifugation at 11,800 rpm for 2 min, followed by resuspension in 500 µl lysis buffer by vortexing. After centrifugation at 7300 rpm for 5 min, the nuclear fraction was resuspended in HBC buffer (Chodavarapu et al., 2010 (link)). The chromatin was digested with a final concentration of 0.02 units/μl MNase (Takara). For primer sequences see Supplemental file 1.