GSM4662814: 10d_Male_H3K27me3_3; Drosophila melanogaster; ChIP-Seq
Adult heads were separated from their bodies by vortexing and sifted through mesh sieves #25 and #40. Adult heads or whole body pupae were dounce homogenized in buffer A1 (60mM KCL, 15mM NaCl, 4mM MgCl2, 15mM HEPES (pH 7.6), 0.5% TritonX-100, 0.5mM DTT, 10mM sodium butyrate, and Roche Protease Inhibitor cocktail) containing 1% formaldehyde solution (Pierce) for crosslinking at RT for 10 min. The crosslinking reaction was quenched using 2.5M Glycine and incubated for 5 min. at RT. Cross-linked homogenates were centrifuged at 4000g for 5 min. at 4ºC, and the nuclear pellets were resuspended in buffer A1 and centrifuged at 4000g for 5 min. at 4ºC. The nuclear pellet was washed by resuspension in buffer A1 and centrifuged as described above two more times and then resuspended in 1x Lysis Buffer without SDS (140mM NaCl, 15mM HEPES (pH 7.6), 1mM EDTA (pH 8.0), 0.5mM EGTA, 1% TritonX-100, 0.5mM DTT, 0.1% sodium deoxycholate, 10mM sodium butyrate, and Roche protease inhibitor cocktail) to remove all traces of A1 buffer before the lysis step. The washed nuclear pellet was resuspended in Lysis buffer containing 1% SDS and 0.5% N-Laurosylsarcosine and incubated at 4ºC, rotating for 1 hr. Nuclear lysates were quickly spun down for 10 seconds at 2000g and supernatant transferred to sonication tubes (Covaris #520130) for shearing. Chromatin was sheared for 25 min. using a Covaris E220 (duty cycle: 5.0, peak incident power: 140, cycles per burst: 200). The sheared chromatin was transferred to microcentrifuge tubes and rotated for 10 min. at 4ºC and then centrifuged at 18,000g for 5 min. at 16 ºC. Sheared chromatin was incubated with 40ul of magnetic protein A/G beads (Pierce) and 5µg of respective antibody at 4ºC overnight. Magnetic beads were washed four times with lysis buffer wash (140mM NaCl, 15mM HEPES (pH 7.6), 1mM EDTA (pH 8.0), 0.5mM EGTA, 1% TritonX-100, 0.05% SDS, 0.5mM DTT, 0.1% sodium deoxycholate, 10mM sodium butyrate, and Roche protease inhibitor cocktail) and twice with TE (10mM Tris-HCL (pH 8.0), 1mM EDTA) for 10 minutes each, rotating at 4 ºC. Beads were incubated in ChIP elution buffer (100mM NaHCO3, 80mM NaOH, 1% SDS) at 37ºC, rotating for 30 min. to elute chromatin. The eluate was diluted 1:10 with Lysis buffer without SDS and incubated on streptavidin beads (Invitrogen Dynabeads C1) for 4hrs rotating at 4ºC. The beads were washed as previous and chromatin reverse crosslinked on beads in ChIP elution buffer (with 0.04mg/ml proteinase K, 0.01mg/ml RNaseA) at 65 ºC overnight. DNA was purified with a DNA purification and concentration kit (Machery-Nagel). ChIPseq libraries were prepared using the NEBNext ChIP-seq library preparation kit. Libraries were size selected using AMPure XP beads (Beckman) and clean up steps were performed using the Machery Nagel PCR cleanup kit. A final wash was performed using the AMpure XP beads according to kit protocol to remove primers.
GEO Accession
GSM4662814