GSM3934392: FASTED-H3K27ac_S2_ChIP-seq II; Mus musculus; ChIP-Seq
Chromatin immunoprecipitations were performed using a previously published protocol with modifications for SI crypts. Briefly, whole isolated crypts from 4 animals were pooled per treatment per replicate and cross linked for 10 min at 37°C with 1% formaldehyde in supplemented media and quenched with 0.125M glycine for 5 min at 37°C. Crypts were washed with PBS with 1× protease inhibitor cocktail (Sigma-Aldrich) and stored at -80°C. Pellets were thawed and then lysed for 30 min on ice with I-ChIP buffer (12mM Tris–HCl pH 8.0, 6mM EDTA pH 8.0, 0.1x PBS, 0.5% SDS) plus cOmplete mini protease inhibitors. Sonication conditions were optimized for SI crypt cells using a Bioruptor (Diagnode) to achieve shear length of 250-500bp. 10% total chromatin was reserved as an input control. Chromatin was diluted 5 fold and immunoprecipitation was performed overnight by incubation of the sonicated cell lysate with 30ul of protein G magnetic dynabeads (Invitrogen) previously coupled to target antibody for a minimum of 1h at 4°C. Immune complexes were then washed five times with cold RIPA buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 140mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC), twice with cold high-salt RIPA buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 500mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC), and twice with cold LiCl buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC). Elution and reverse cross linking was performed in 50ul direct elution buffer (10mM Tris–HCl, pH 8.0, 5mM EDTA, pH 8.0, 300mM NaCl and 0.5% SDS) with Proteinase K and RNaseA at 65°C overnight. Eluted DNA was cleaned up with solid-phase reversible immobilization (SPRI) beads (Beckman-Coulter). Libraries for Illumina sequencing were generated following the New England BioLabs NEBNext Ultra DNA Library Prep Kit protocol. A total of 10x cycles were used during PCR amplification for the generation of all ChIP-seq libraries. Amplified ChIP DNA was purified using double-sided AMPure XP to retain fragments ~200 to 500bp and quantified using the Qubit 2000 and TapeStation 2000 before multiplexing.