CENH3 CHiP-seq of wheat cultivars
Chromatin immunoprecipitation (ChIP) was performed according to the method described by Nagaki et al. (2003) with slight modification using wheat CenH3 antibody (Koo et al., 2015). Nuclei were isolated from 2-week-old seedlings and digested with micrococcal nuclease (Sigma-Aldrich, St. Louis, MO) to liberate nucleosomes. The digested mixture were incubated overnight with 3 g of antibody at 4C. The target antibodies were captured from the mixture using Dynabeads Protein G (Invitrogen, Carlsbad, CA). In mock experiment, the antibody was substituted with rabbit serum, which serves as nonspecific binding control in chromatin immunoprecipitation assay. To start chromatin elution, the chromatin elution buffer (1% sodium dodecyl sulfate, 0.1 M NaHCO3) was preheated at 65C for 30 min and the same (100 l) was used to elute the chromatins. DNA isolation from chromatin immunoprecipitated and mock control samples were done using ChIP DNA Clean & Concentrator Kit (Zymo Research, Tustin, CA). The ChIP-seq libraries were constructed using TruSeq ChIP Library Preparation Kit (Illumina, San Diego, CA) following the manufacturers instructions and the libraries were sequenced in NovoSeq S4 with 2x150bp sequencing run. Nagaki K, Talbert PB, Zhong CX, Dawe RK, Henikoff S, Jiang J (2003) Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres. Genetics 163: 12211225. Koo DH, Sehgal SK, Friebe B, Gill BS (2015) Structure and stability of telocentric chromosomes in wheat. PLoS One 10: e0137747.