Comparison of ChIP of H3K27ac in control wing imaginal discs and in salm/salr knock down wing imaginal discs
Comparison of ChIP of H3K27ac in control wing imaginal discs and in salm/salr knock down wing imaginal discs
We used 120 imaginal discs were per sample.  Discs were fixed in 1.8 % formaldehyde, snap frozen and store at -80 until required. 100 frozen disks were thawed on ice and 500 mL RIPA buffer (140mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 1x Roche cOmplete Protease inhibitors) was added. The samples were then sonicated in the Bioruptor Pico using 1.5 ml sonication tubes (Diagenode C30010016) according to the manufacturer's instructions for 12 cycles (30 sec on / 30 sec off). The supernatant was transferred into 1.5ml low binding tubes (Eppendorf, 0030108051) and centrifuged at 20.000 xg for 10 min at 4C. The next day the DNA was purified with Phenol-Chloroform purification and precipitated with ethanol, Sodium Acetate pH 5.3 and glycogen to obtain pure DNA. Thaw chromatin on ice. 1 microl of H3K27ac antibody (Histone antibody anti K27ac ab4729), was incubated overnight with the chromatin in RIPA buffer in a total volume of 900 ml. The next day 25 ml of magnetic protein A/G beads (Dynabeads, Invitrogen, 10002D and 10004D) were washed with 1ml of RIPA buffer and added to the IPs for an additional 3 hour incubation on the rotating wheel at 4C.  The ChIPs were then washed for 10 min on the rotating wheel. The chromatin was then RNase-treated and reverse cross-linked as described for the quality check of the chromatin. ChIP-seq libraries were prepared following the manufacturer's manual of NEBNext Ultra II DNA Library Prep Kit for Illumina E7645S