mammary gland/breast; derived from metastatic site: pleural effusion
Sequenced DNA Library
Cells were lysed with 0.5% SDS buffer containing protease inhibitors cocktail (SIGMA, catalog# P8340), scraped and centrifuged at 1150 g for 10 min at 4⁰C. Pellets were resuspended in 4 ml of ice-cold IP Buffer composed by a 2:1 micture of SDS buffer (100 mM NaCl, 50mM Tris-Cl, pH8.1 EDTA, pH 8.0, 0.5% SDS) and Triton Dilution Buffer (100 mM Tris-Cl, pH 8.6, 100 mM NaCl, 5mM EDTA, pH 8.0, 5% Triton X-100) containing protease inhibitor cocktail. Samples were sonicated with Branson Digital Sonifier (Danbury, USA) in 30s bursts followed by 30s of cooling on ice for a total sonication time of six minutes per sample. Chromatin was pre-cleared for 1 h at 4 ⁰C with Sepharose protein G beads (Life technologies, catalog# 101242) and subsequently precipitated overnight at 4 ⁰C with 4 µg of either anti-BMAL1 (Protein Tech, catalog# 14268-1-AP) or 4 µg of anti-MAX (Bethyl Lab, catalog#A302-866A). DNA protein complexes were recovered with Sepharose protein G beads for 3 h and washed twice sequentially with Mixed Micelle Wash Buffer (150 mM NaCl, 20 mM Tris-Cl, pH 8, 5 mM EDTA, 5% w/v sucrose, 1% Triton X-100, 0.2% SDS), LiCl/Detergent buffer (0.5% Na-deoxycholate, 1 mM EDTA, 250 mM LiCl, 0.5% (v/v) NP-40, 10 mM Tris-Cl, pH 8.0), Buffer 500 (0.1% (w/v) deoxycholic acid, 1 mM EDTA, 50 mM HEPES, pH 7.5, 500 mM NaCl, 1% (v/v) Triton X-100) and TE buffer (10mM Tris-Cl, 1mM EDTA, pH 8.0). Beads were further supended in TE-S buffer (TE buffer, 2% SDS) and treated with RNAse A for 30 min at 37 ⁰C. Cross-linking was reverted by overnight incubation at 65 ⁰C in TE-S containing 0.4 mg/ml proteinase K. In parallel, inputs were treated in the same way. Immunoprecipitated and input DNA was purified using PCR purification kit (QIAGEN, catalog#28106) using 60 µl of buffer T (10mM Tris-Cl, pH 8.0). Input and immunoprecipitated DNA (1–10 ng) were blunt-ended and phosphorylated, and a single 'A' nucleotide was added to the 3' ends of the fragments in preparation for ligation to an adapter that has a single-base 'T' overhang. The ligation products were purified and accurately size-selected by agencourt AMPure XP beads. Purified DNA was PCR-amplified to enrich for fragments that have adapters on both ends. All steps were on automation instrument, Biomek FX by Beckman Coulter. The final purified product was then quantitated prior to cluster generation on bioanalyzer 2100. Libraries with distinct adapter indexes were multiplexed (1/5 libraries per lane) and after cluster generation on FlowCell were sequenced for 50 bases in the single read mode on a HiSeq 2000 sequencer.