Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Approximately 1x107 H9 hESCs were used to make each nasChIP-seq library. Anti-CTCF (Millipore) or -SMC1A (Millipore) antibodies were pre-incubated with Dynabeads Protein A beads (Thermo Fisher). Extracted chromatin was incubated with antibody-beads complex on a rotator at 4 °C for 5 hr. Beads were washed once with Wash Buffer (20 mM Tris-HCl pH 8.1, 500 mM NaCl, 2 mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS), twice with LiCl Buffer (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA pH 8.0, 1% NP-40, 1% sodium deoxycholate), and once with 1x TE pH 8.0 with 50 mM NaCl. DNA was eluted by incubating beads in Elution Buffer at 65 °C for 30 min. Crosslinking-reversal, proteinase K digestion, phenol:chloroform:isoamyl alcohol extraction, ethanol precipitation, click chemistry, ethanol precipitation, streptavidin beads pull-down, and Illumina library preparation steps were the same as described above for nasBS-seq.