CENH3 ChIP-seq of Rosa canina
Young leaves (around 2-5 g) of R. canina were collected and crosslinked in 4% formaldehyde in 1 PBS on ice with vacuum infiltration applied for 30 min. The quenching was performed by applying 1M Glycine in each sample followed by vacuum infiltration at room temperature for 15 min. The material was then macerated in liquid nitrogen and the chromatin was extracted. After extraction, the chromatin was sonicated for 30 min on a Bioruptor (Diagenode) until achieving fragments around 200-600 bp. The sonicated chromatin was incubated overnight at 4 C with 1 g of each polyclonal antibody. Samples with no addition of primary antibodies were also incubated as input control samples and at least two experimental replications were used for each ChIP combination. After incubation, protein beads were washed and added to each complex protein-antibody and incubated for at least 2 h at 4 C in slow rotation. The final recovered chromatin was eluted from the beads, followed by a de-crosslink step and final DNA extraction.