Anti rad21 ab

ChIP-seq libraries were prepared from two biological replicates as previously described (3 (link)). Briefly, ∼10 M cells were formaldehyde-fixed for 10 min, scraped into lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 5 mM MgCl₂, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, cOmplete protease inhibitor; Roche) and nuclei were isolated in SDS lysis buffer (1% sodium dodecyl sulphate (SDS), 10 mM ethylenediaminetetraacetic acid, 50 mM Tris-HCl pH 8.1, 1 mM PMSF, cOmplete protease inhibitor; Roche). Cleared lysates were sonicated for 26 cycles using Bioruptor Next-Gen (Diagenode). RAD21-bound DNA complexes were purified using 5 μg of Anti-Rad21 Ab (ab992, Abcam), anti-H3K9me3 Ab (Active Motif, #39161) and anti-H3K27me3 (Millipore #07–449). Library preparation continued as described (3 (link)), products were amplified using 15–20 cycles, fragments were size-selected (230–350 bp) from Novex 10% Tris/Borate/EDTA (TBE) gels (Life Technologies, Carlsbad, CA, USA) and libraries were sequenced using Illumina HiSeq 2000 at EMBL Genomics Core Facility (Heidelberg, Germany).