GSM5941870: H2B8 anti-GFP ChIP-seq rep 2 (PBS control); Arabidopsis thaliana; ChIP-Seq
Ten-day-old seedlings of a p35S::H2B.8-eGFP line were ground with a pestle and mortar in liquid N2 and homogenized in nuclei isolation buffer (0.25 M sucrose, 15 mM PIPES pH 6.8, 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, 1 mM PMSF, 1x proteinase inhibitor Cocktail). Nuclei were separated from debris by filtering through two layers of miracloth. Chromatin was fragmented by micrococcal nuclease (MNase, New England Biolabs) digestion. For IP samples, fragmented chromatin was immunoprecipitated with GFP-Trap beads. For input, no immunoprecipitation was undertaken. Protein and RNA was removed by digestion with Proteinase K and RNase A, respectively. Phenol-chloroform extraction was used to obtain DNA. Libraries were prepared using the Ovation Ultralow System V2 (Nugen, 0344) and sequenced on the NextSeq 500 (Illumina) with 76 bp single end reads at the John Innes Centre, Norwich, UK.