The chromatin immunoprecipitations and subsequent deep sequencing in Figures 5 and 6 were also performed according to previous reports (Shang et al., 2010 (link), 2013 (link)). The MNase (Takara, Kyoto, Japan)-digested chromatin of 1.5 × 109 cells was extracted with 0.5 M NaCl–containing buffer, and the extract was exposed for 4 h at 4°C to Sepharose-Protein G beads preincubated with rabbit anti–CENP-A or -H3K9me3 antibodies (MBL, Nagoya, Japan). Beads were washed, and the bound DNA was purified and applied to deep sequencing.
ChIP-seq libraries were constructed as described in the Illumina TruSeq DNA LT Sample Prep Kit protocols. Briefly, ∼50 ng of purified DNAs were end repaired, followed by the 3′ addition of a single adenosine nucleotide and ligation to universal library adapters. DNA libraries were prepared by eight cycles of PCR amplification. ChIP DNA libraries were sequenced using the Illumina HiSeq 2500. Sequencing of libraries was performed up to 2 × 151 cycles. Image analysis and base calling were performed with the standard Illumina pipeline version RTA1.17.21.3.
Sequencing data were mapped to chicken genome database galGAL4 (UCSC Genome Browser) with a BWA 0.6.2 mapping tool (Li and Durbin, 2009 (link)). A 4% mismatch was allowed for the mapping.Figures 5 and 6 represent raw sequence reads. Input data are also shown.
ChIP-seq libraries were constructed as described in the Illumina TruSeq DNA LT Sample Prep Kit protocols. Briefly, ∼50 ng of purified DNAs were end repaired, followed by the 3′ addition of a single adenosine nucleotide and ligation to universal library adapters. DNA libraries were prepared by eight cycles of PCR amplification. ChIP DNA libraries were sequenced using the Illumina HiSeq 2500. Sequencing of libraries was performed up to 2 × 151 cycles. Image analysis and base calling were performed with the standard Illumina pipeline version RTA1.17.21.3.
Sequencing data were mapped to chicken genome database galGAL4 (UCSC Genome Browser) with a BWA 0.6.2 mapping tool (Li and Durbin, 2009 (link)). A 4% mismatch was allowed for the mapping.
