GSM8774979: Lj8627 ChIP CENH3 2; Lemna japonica; ChIP-Seq
Chromatin extraction and immunoprecipitation was performed as previously described (Parent et al. 2021) with minor modifications. Briefly, two biological replicates of 750 mg whole frond tissue per clone were crosslinked in 1% formaldehyde for 10 min, and ground to a fine powder under liquid nitrogen. Nuclei were extracted in an SDS-Tris buffer, lysed, and sonicated with a Bioruptor Pico (Diagenode) for 12 cycles at 30 Hz, 30 s on, 30 s off. CENH3 orthologues in S. polyrhiza, W. australiana, L. gibba, and L. japonica were identified by sequence similarity search with O. sativa and A. thaliana sequences. A rabbit polyclonal antibody was raised against the synthesized polypeptide sequence “RTKHISGKRRRTE” unique to the L. minor and L. turionifera CENH3 N-terminal tail (LjCENH3), and then validated by Western blot. Chromatin was cleared with Protein A magnetic beads (Thermo Fisher Scientific) and incubated overnight with following antibodies: LjCENH3 (GenScript), H3 (Sigma, H0164), and H3K4me3 (Sigma, 07-473). Immune complexes were eluted, and crosslinks were reversed with Proteinase K digestion for 2 hrs at 55C with gentle shaking. Overnight NaCl incubation was omitted as in 129. DNA was purified with the ChIP Purification Kit (Zymo), and the NEBNext Ultra II DNA library prep kit (New England Biolabs) was used to construct sequencing libraries. L. turionifera samples in these libraries were from the clone 8133, rather than 9434.