Frozen-stored mammary tissues collected at lactation day 1 (L1) were ground into powder. Chromatin was fixed with formaldehyde (final 1%) for 15 min at room temperature, and then was quenched with glycine (final 0.125 M). Pellets were lysed in lysis buffer (10 mM Tris HCI pH 8.0, 10 mM NaCl, 0.5% Nonidet P-40) containing Phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors, incubated on ice for 30 min, and dounced using pre-chilled glass homogenizer, followed by another 10 min incubation on ice. After removal of supernatant, nuclei pellets were re-suspended in restriction endonuclease buffer 2 (NEB; 50 mM NaCl,10 mM Tris-HCl,10 mM MgCl2,1 mM Dithiothreitol; New Englnad Biolabs). SDS was added to a final concentration of 0.2% and samples was incubated for 1 hr at 37°C. 2% Triton X-100 was added, followed incubation for 1 hr at 37°C. Samples were incubated with 400 units each of restriction enzymes BamHI (New Englnad Biolabs) and were incubated overnight at 37°C. To enzyme inactivation SDS was added (final 1%) and incubated for 30 min at 65°C, followed by SDS-sequestration with 1% Triton X-100 at 37°C for 1 hr, T4 ligase (New England Biolabs) was added to each sample and incubated for 1 hr at 16°C. Ligated samples were treated overnight with proteinase K (20 mg/mL, Invitrogen) at 65°C and 1 hr at 37°C with RNase A (10 mg/mL, Thermo Fisher Scientific), and DNA fragments were purified by phenol-chloroform method. DNA fragments from 3C were digested with NlaIII (New England Biolabs) overnight at 37°C and ligated using T4 ligase overnight at 16°C. DNA was purified by phenol/chloroform method and then amplified with site-specific primers linked to the Illumina DNA adaptors. Libraries for next generation sequencing were prepared and sequenced with HiSeq 2500 (Illumina)
GEO Accession
GSM2525546