GSM8796172: NE_Ctrl_ChIP_Input_Rep2; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde in 1x PBS for 10 min at room temperature. The crosslinking reaction was quenched by adding 0.125 M glycine for 5 min at room temperature. Crosslinked cells were centrifuged at 600 x g for 5 min at 4°C, and the supernatant was removed. The cell pellet was washed with ice-cold 1x PBS, centrifuged again at 600 x g for 5 min at 4°C, and the supernatant was aspirated. This wash step was repeated once more. The final cell pellet was frozen on dry ice and stored at -80°C until use.  Crosslinked human cells were resuspended in Lysis Buffer1 (10 mM Tris-HCl [pH 8.0], 10 mM NaCl, 0.5% NP-40, and 1x Complete Protease Inhibitor EDTA-free) and incubated on ice for 10 min. For Drosophila S2 spike-in controls, crosslinked S2 cells were resuspended in 100 uL Lysis Buffer1 at a final concentration of 2.0x105 or 4.0x105 cells/uL, and 25% of the S2 cells were mixed with human cells. The mixed cell suspension was centrifuged at 665 x g for 5 min at 4℃, and the supernatant was removed. The cell pellet was then resuspended in Lysis Buffer2 (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 0.32% SDS, 1x Complete Protease Inhibitor EDTA-free) and incubated on ice for 10 min. The cell lysate was then diluted in IP Dilution Buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 1x Complete Protease Inhibitor EDTA-free) and transferred to a milliTUBE ATA Fiber (Covaris). Chromatin was sonicated using a Covaris S220 (Covaris) for 5.5 min. Insoluble debris was removed by centrifugation at 12,000 x g for 5 min at 4℃. The supernatant containing sonicated chromatin was transferred to a new tube and stored at -80℃ until use.  Dynabeads Protein-G (10004D, Thermo Fisher Scientific) (25 - 80 uL) were washed twice with PBS-T (0.02% Tween-20 in 1x PBS). The desired amounts of H3K9me3 (ab8898, Abcam) antibodies were conjugated to washed Dynabeads by incubating for 2 to 6 hours at 4°C with rotation. Meanwhile, frozen sonicated chromatin was thawed on ice. A desired amount of chromatin (Table S3, based on DNA quantity) was diluted with IP dilution buffer at a 4:1 ratio. Antibody-conjugated Dynabeads were washed twice with IP Dilution Buffer (without protease inhibitor), resuspended in the diluted chromatin, and incubated overnight at 4°C with rotation. The next day, ChIPed beads were washed four times using the following washing buffers: (1) FA Lysis Buffer (50 mM HEPES-KOH [pH 7.5], 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.1% Sodium deoxycholate, 1x Complete protease inhibitor EDTA-free); (2) NaCl Buffer (50 mM HEPES-KOH [pH 7.5], 500 mM NaCl, 2 mM EDTA, 1% TritonX-100, 0.1% Sodium deoxycholate); (3) LiCl Buffer (100 mM Tris-HCl [pH 8.0], 500 mM LiCl, 1% NP-40, 1% Sodium deoxycholate); (4) 10 mM Tris-HCl [pH 8.0]. Following washing steps, chromatin was eluted with TES buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS) for 15 min at 65℃ while shaking at 1,000 rpm in ThermoMixer F1.5 (Eppendorf). The ChIP and Input chromatin were reverse-crosslinked by adding NaCl to a final concentration of 200 mM and incubating for 8 hours at 65°C. Samples were then treated with 50 ng/uL RNaseA (EN0531, ThermoFisher Scientific) for 30 min at 37°C, followed by treatment with 0.2 mg/mL Protease K (03115828001, Roche) for 2 hours at 37°C. DNA was purified using phenol-chloroform extraction, followed by ethanol precipitation. DNA concentration was measured using Quantus fluorometer (Promega). Multiplexed libraries were prepared from ChIP and Input samples (two replicates) using NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, NEB), following the manufacturer's protocol with the following modifications. After adaptor ligation, cleanup was performed using AMPure XP Beads (A63880, Beckman Coulter), MagBind Magnetic Beads (M1378-01, Omega), or homemade Sera-mag beads (Rohland et al., Genome Res. 2012) without size selection. After RCR enrichment of the adaptor-ligated library, two rounds of size selection were performed using the AMPure XP Beads or MagBind Magnetic Beads.