cells growing asynchronously were crosslinked (10 min , 1 % formaldehyde, 23˚C) quenched for 10 min by 200 mM glycine, washed three times with PBS, and then resuspended in chromatin buffer containing 150 mM NaCl, 1% Triton X100, 0.1% SDS, 20 mM Tris HCl pH8.0, and 2 mM EDTA. DNA was sheared using Covaris S220, so that most fragments were in the 300-500 bp range. Chromatin was precipitated overnight with magnetic beads (DiaMag, Diagenode, Inc.) loaded with anti-CTCF or anti-BORIS antibodies. The precipitate was washed, crosslinks reversed, protein component was digested with proteinase K, and DNA was extracted using phenol/chloroform/isoamyl alcohol. DNA concentration was measured by Qubit (Life Technologies) and/or Nanodrop (Thermo Scientific) fluorimeters. The immunoprecipitated DNA was amplified using the Phi29 strand-displacement procedure (GE Bioscience) following the concatemerization of precipitated DNA fragments via ligation to double strand adaptors containing BamHI overhangs and internal SapI sites. Both amplified and nonamplified samples showed essentially the same relative enrichment for known sites of CTCF and BORIS binding. Following the amplification, adapters were removed by SapI digestion and agarose gel purification. Input DNA was used as a hybridization reference for the hybridization of amplified ChIP DNA
