title: H3K9me3 ChIP-seq from Drosophila simulans: ovaries, cold condition
design: Chromatin immunoprecipitation was performed using 50 ovary pairs dissected from three to five day old females. Ovaries were re-suspended in A1 buffer containing 60mM KCl, 15mM NaCl, 15mM Hepes, 0,5% Triton and 10mM Sodium butyrate. Formaldehyde (Sigma) was added to a final concentration of 1.8% for secondary cross-linking for 10 min at room temperature. Formaldehyde was quenched using glycine (0.125 M). Cross-linked cells were washed and pelleted twice with buffer A1, once with cell lysis buffer (140mM NaCl, 15mM Hepes, 1mM EDTA, 0.5mM EGTA, 1% Triton X100, 0.1% Sodium deoxycholate, 10mM sodium butyrate) , followed by lysis in buffer containing ,140mM NaCl, 15mM Hepes, 1mM EDTA, 0.5mMEGTA, 1% Triton X100, 0.5% SDS, 0.5% N-Laurosylsarcosine, 0.1% sodium deoxycholate, 10mM sodium butyrate for 120 min at 4C. Lysates were sonicated in Bioruptor sonicator to reach a fragment size window of 200-600 bp. Chromatin was incubated overnight at 4C with the following antibodies: for H3K9me3 ChIP using -H3K9me3 (actif motif #39161, 3g/IP) and for H3K4me3 using -H3K4me3 (millipore #07-473, 3g/IP) antibodies. The Magna ChIP A/G Chromatin Immunoprecipitation Kit  (cat# 17-10085) was used following manufacturers instructions. Final DNA recovery was perfomed by classic phenol/chloroform DNA precipitation method using MAxtract high density tubes to maximize DNA recovery.