GSM8783780: BHLHE40_DEC1; Mus musculus; ChIP-Seq
FACS-purified fixed cells (typically ~1million) were pelleted and flash-frozen. ChIP-seq was conducted as described in (Gifford et al., 2013) with minor modifications. Briefly, cell pellets were thawed on ice for 30min, incubated in lysis buffer (0.5% NP-40 +85mM KCl +20mM Tris-HCl pH8.0 +protease inhibitor) for 10min on ice, and nuclei were pelleted and incubated in lysis buffer (1% NP-40 +0.5% sodium deoxycholate +0.1% SDS +10mM Tris-HCl pH7.5 +protease inhibitor) for 10min on ice. Chromatin was then sheared with a Branson Sonifier (model S-450D) at 4ºC and incubated with 1µg/million cells H3K27Ac (Active Motif; 39133) or H3K4me1 (Millipore, 17-614) antibody overnight at 4ºC. Next, antibody-protein complexes were isolated by incubation with Protein A/G beads (Life Technologies; 100-02D/100-07D) for 2h at 4ºC. Samples were then sequentially washed twice with low-salt buffer (0.1% SDS +1% Triton X-100 +2mM EDTA +20mM Tris-HCl pH8.1 +150mM NaCl), twice with high-salt buffer (0.1% SDS +1% Triton X-100 +2mM EDTA +20mM Tris), twice with LiCl buffer (0.25M LiCl +1% NP-40 +1% deoxycholate +1mM EDTA +10mM Tris-HCl pH8.1), twice with TE (10mM Tris-HCl pH8.0 +1mM EDTA), and finally eluted in freshly-prepared elution buffer (1% SDS +0.1M NaHCO3) at 65ºC for 30min. Eluates were then treated with reverse crosslinking salt mixture (250mM Tris-HCl pH6.5 +62.5mM EDTA pH8.0 +1.25M NaCl +5mg/ml Proteinase K + 62.5ng/µl RNase A) overnight at 65ºC DNA was purified using AMPure XP magnetic beads (Beckman Coulter), and sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs; E7103), pooled, and sequenced on a HiSeq 2500 instrument (Illumina).