J180-3 ChIP-seq
Whole seedlings between 3 and 13 g in weight, including roots, were harvested and frozen in liquid nitrogen, then finely ground with pre-chilled mortars and pestles. Between 3 and 4 g of each were used for ChIP using a native ChIP protocol with micrococcal nuclease digest of the DNA. Rice CENH3 polyclonal antibody with broad reactivity to grass CENH3 was used (Nagaki et al., 2004, Nature Genetics). For each ChIP and input sample, 5-30 ng of DNA was used for preparing Illumina sequencing libraries (KAPA hyper prep kit #KK8500). Barcoded adapters were used for pooling libraries (Bioo Scientific NEXTflex™ Bisulfite-Seq Barcodes, #511912). Libraries were amplified with 5 or 6 cycles of PCR, and amplicons of 100-200 bp were separated from longer fragments by gel electrophoresis and purified without heating (Qiagen QIAquick Gel Extraction Kit #28704)