title: GSM4838451: P17_MEL1_D13_TNT_H3K9me3_ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were crosslinked for 10 minutes in 1% formaldehyde and quenched in 125 mM glycine. Prior to ChIP, antibodies were bound to beads by mixing 3 µg H3K9me3 antibody (Abcam, ab8898) with 50 µl washed Dynabead M-280 Sheep Anti-Rabbit IgG  (Thermofisher) in 500 µl RIPA-150 buffer (50 mM Tris-HCl pH 8.0, 0.15 M NaCl, 1 mM EDTA , 0.1% SDS, 1% Triton X-100 and 0.1% sodium deoxycholate) and incubated at 40C for 6 hours on a rotator . Crosslinked cells were lysed on ice for 10 min in 15 ml ChIP lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) supplemented with 1x EDTA-free Protease Inhibitor Cocktail (Roche). Lysed cells were centrifuged at 3,200xg for 5 min, supernatant removed and followed by two washes with 10ml ChIP wash buffer (10 mM Tris-Cl pH 8.0, 200 mM NaCl and 1 mM EDTA pH 8.0). Lysed cells were resuspended in 130 µl nuclei lysis buffer (50mM  Tris-HCl pH 8.0, 10mM EDTA and 1% SDS) supplemented with 1x EDTA-free Protease Inhibitor Cocktail (Roche), transferred to Covaris tubes (microTUBE AFA Fiber 6x16mm) and sheared with the Covaris (S220) for 5 min (5% duty cycle, 200 cycles per burst and 140 watts peak output at 40C). Sheared chromatin was transferred to 1.5 ml eppendorf tubes, centrifuged at 10,000xg for 10 min. The supernatant was transferred to 2 ml low-bind tubes containing 1.2 ml ChIP dilution Buffer (50 mM Tris-HCl pH 8.0, 0.167 M NaCl, 1.1% Triton X-100 and 0.11% sodium deoxycholate) and 0.65 ml RIPA-150 buffer, and incubated with the previously prepared H3K9me3 antibody bound Dynabeads at 40C overnight on a rotator. Chromatin bound beads were subsequently washed one time with 1 ml RIPA-150 buffer, two times with 1 ml RIPA-500 buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 1 mM EDTA , 0.1% SDS, 1% Triton X-100 and 0.1% sodium deoxycholate), two times with 1ml RIPA-LiCl buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA , 1% NP-40, 0.7% sodium deoxycholate and 0.5 M LiCl2) and two times with TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). After wash steps, DNA was eluted, crosslinks were reversed, and immunoprecipitated DNA was purified by Agencourt AMPure XP beads (Beckman Coulter, Cat no: A63880). Libraries were prepared from ChIP eluate containing 10 ng DNA using the SMARTer ThruPLEX DNA-Seq Kit (Takara, R400675) with SMARTer DNA unique dual index (Takara, R400665). After limited PCR amplification, libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Cat no: A63880), and eluted in a final volume of 20 ul. Libraries were sequenced on the Novaseq 6000.