Chromatin immunoprecipitation (ChIP) and ChIP-seq

ChIP analysis was performed essentially as described previously^37,38. In brief, cells were cross-linked with 1% formaldehyde for 10 min and stopped with 125 mM glycine. The isolated nuclei were resuspended in nuclei lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS) and sonicated using a Bioruptor Sonicator (Diagenode). The samples were immunoprecipitated with 2–4 μg of the appropriate antibodies overnight at 4 °C. Protein A/G beads (Millipore) were added and incubated for 1 hour, and the immunoprecipitates were washed twice, each with low-salt (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 0.1% SDS), high-salt (20 mM Tris pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 0.1% SDS) and LiCl buffer (20 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40 and 1% SDS). Eluted DNA was reverse-crosslinked, purified using PCR purification kit (Qiagen), and analyzed by quantitative real-time PCR on the ABI 7500-FAST System using the Power SYBR Green PCR Master Mix (Applied Biosystems).

For ChIP-seq, the ChIP experiments were carried out essentially the same as described above. Purified DNA was sequenced using the Illumina Solexa Hiseq 3000. The raw reads were mapped to human reference genome NCBI 37 (hg19) or the Drosophila melanogaster genome (dm3) by bowtie v1.1.0, allowing up to 1 mismatch. Only uniquely mapped reads were retained for peak calling. But before that, we use spike-in normalization for sample size correction as previously described³⁹. For simplicity, the reads were downsampled to keep same spike-in reads count in different samples. Then the ChIP-seq peaks were called by MACS v1.4.2 with a cutoff of p ≤1e−8, and clonal reads were automatically removed by MACS. The ChIP-seq reads density was determined by deepTools v2.3.4, and then the average binding profile and heatmap were visualized using R v3.2.3 (Supplementary Tables 1–4).