GSM5411927: GF_H4ac4_rep1; Mus musculus; ChIP-Seq
To prepare epithelial cell pellets, colons were incubated in PBS containing 30 mM EDTA and 1.5 mM DTT for 20 minutes at 4°C with occasional inversion.  Tissue was subsequently transferred to PBS containing 30 mM EDTA, incubated for 10 mins at 37°C, and shaken vigorously for 1-2 minutes until the mesenchyme separated from the epithelium.  The mesenchyme was removed and epithelial cells were pelleted at 1500 rpm for 5 minutes at 4°C and washed in PBS.  Cell pellets were further incubated with dispase (Corning, 0.3 U/ml) in PBS (100 l dispase per 50 ml PBS) at 37°C for 10 mins and then quenched with 1/10 volume of FBS containing DNase followed by washing in PBS.  Cell pellets were flash frozen in liquid nitrogen and stored at -80°C until use.  For ChIP-seq, cells were cross-linked prior to freezing by treatment with 1% formaldehyde in PBS for 5 mins at room temperature, followed by quenching with 125 mM glycine for an additional 5 mins at room temperature.  Cross-linked pellets were then washed in PBS and flash frozen until use.    Nuclear lysates were prepared by resuspending cells in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x protease inhibitors) and rotating for 10 mins at 4°C.  After centrifugation at 3000 rpm for 5 mins at 4°C, the cell pellet was resuspended in lysis buffer 2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x protease inhibitors) and rotated for 10 mins at room temperature.  Cells were pelleted and then resuspended in lysis buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mm EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.1% N-lauroylsarcosine, 1x protease inhibitors) for chromatin shearing with a Covaris S220 focused ultrasonicator (8.4W, 200 peak power, 200 cycles/burst, 900 seconds) in a 4°C water bath.  After sonication, Triton X-100 was added to 1% final concentration and the lysate was cleared by centrifugation at top speed in a microfuge for 10 mins at 4°C.  Magnetic protein G beads were washed with blocking solution (0.5% BSA in PBS) and then incubated with rabbit anti-serum against acetylated histone H4 (Millipore 06-866) in blocking solution for several hours at 4°C with rotation.  Beads were then washed in blocking solution and then added to nuclear lysates containing a spike-in of Drosophila S2 chromatin (cells generously provided by P. Pascual-Garcia), prepared as above.   After overnight incubation at 4°C, beads with captured chromatin were washed with 3 x 1 ml RIPA wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate) and 1 x 1 ml of final wash buffer (10 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM EDTA).  Beads were eluted with 200 µl elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) for 30 mins at 65°C.  The supernatant was then incubated at 65°C overnight to reverse cross-links.  After dilution with one volume of TE, RNase was added to 0.2 mg/ml and incubated at 37°C for 2 hrs.  Proteinase K was then added to 0.2 mg/ml and incubated at 55°C for 2 hrs.  DNA was purified by phenol-chloroform extraction with subsequent ethanol precipitation. Purified DNA was resuspended in 10 mM Tris pH 8 and prepared for sequencing using the NEBNext Ultra II DNA library kit for Illumina (New England Biolabs).