Genome-wide DNA Methylation Analysis

Genomic DNA was treated using bisulfite, amplified (primers in Table S2), and subcloned. DNA from clones (>10 clones/amplicon) was sequenced as described elsewhere [15] (link).
For co-IP, 6×10⁶ MEL or K562 cells were lysed (RIPA) and gently sonicated. 100 µg of precleared protein extract was incubated 3 hrs/4°C with anti-Ctcf (cat#07–729) or anti-Smarca5 (cat#07–624, Upstate) and next with proteinA/proteinG overnight. Control antibody: IgG, cat.# NI01, Calbiochem, 5∶100). Immunoprecipitates (IP) were washed with set of buffers. IPbuffer (0.02% SDS/2%Trion X-100/4 mM EDTA/40 mM Tris-HCl (pH = 8)/300 mM NaCl), WashI (0.1%SDS/1%Triton X-10/2 mM EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/50 mM NaCl), WashII (0.1% SDS/1% Triton X-10/1% EDTA (pH = 8)/20 mM Tris-HCl (pH = 8)/500 mM NaCl). IPs were resolved on SDS/PAGE, blotted, and immune-detected.
Chromatin immunoprecipitation (ChIP) [19] (link) lysates were controlled for DNA purity&quantity by Nanodrop ND-1000. Antibodies: Smarca5/Snf2h (cat.#ab3749, Abcam, 3 µg/IP), Ctcf (cat.#ab10571, Abcam, 2 µg/IP), RAD21 (cat.#ab992, Abcam, 2 µg/IP), and SMC1 (cat.# ab9262, Abcam, 2 µg/IP). Control IgG: cat.#NI01, Calbiochem, 5∶100.