Spike in chromatin

Lysates of crosslinked cells were sonicated in lysis buffer (10 mM Tris-HCl pH8.1, 150 mM NaCl, 5 mM EDTA, 0.5% Sarkosyl, 0.1% sodium deoxycholate, complete protease inhibitor cocktail, 1 mM NaF, 0.1 mM Na₃VO₄, and 10 mM glycerophosphate) using a Bioruptor Plus (12 cycles of 30s on/off, high power) to obtain DNA fragment averaging 500–700 bp. Soluble chromatin fraction was diluted 1:5 (10 mM Tris-HCl pH8.1, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, complete protease inhibitor cocktail, 1 mM NaF, 0.1 mM Na₃VO₄, and 10 mM glycerophosphate) with the addition of 40 ng Spike-in Chromatin per reaction (Active Motif 53083). Immunoprecipitation was conducted using 4 μg of anti-H3 (Abcam ab1791), 4 μg anti-H3K27me3 (Sigma-Aldrich 07–449), 4 μg anti-H3K4me3 (Sigma-Aldrich 07–473), 4 μg control IgG (Sigma-Aldrich 12–370), 2 μg Spike-in Antibody (AB_2737370) and 900 μg of chromatin at 4°C for 16 h. Immunoprecipitates were incubated with 35 μl of Dynabeads Protein G for 2 h, washed 2x with Low-salt wash buffer (20 mM Tris-HCl pH 8.1, 2 mM EDTA, 200 mM NaCl, 1% Triton X-100), 2x in LiCl buffer (10 mM Tris-HCl pH8.1, 2 mM EDTA, 1% NP40, 250 mM LiCl), and 1x in TE. Chromatin was eluted and purified as in the IRF4 ChIP experiments. Primers were designed according to (Liang et al., 2013 ) (GAPDH) and (Cao et al., 2014 (link)) (MYT1). All primer sequences are indicated in Table S7.

Native and crosslinking (X) ChIP were performed with 5 × 10⁶ and 6 × 10⁷ cells, respectively, as previously described². A spike-in normalization strategy was used to normalize all ChIP-seq data to reduce the effects of technical variation and sample processing bias. Spike-In chromatin (Active Motif, 53083) and Spike-in antibody (Active Motif, 61686) were used according to manufacturer instructions.