GSM8461114: ChIP-seq H3K9me3 library from MORC2 KO HEK293T cell line - Replicate 1; Homo sapiens; ChIP-Seq
5-10 x 106 cells per IP were cross-linked by adding 1/10 volume of fresh 11% formaldehyde solution (50mM HEPES-KOH pH 7.4, mM NaCl, 1 mM EDTA, 1 mM EGTA, 11% formaldehyde) to a 1 x 106 cell suspension for 20 minutes at room temperature with mixing. 1/12.5 volumes of 2.5 M glycine was added to quench formaldehyde and cells were washed twice in ice-cold PBS. Cross-linked cells were lysed in Lysis Buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) IGEPAL CA-630, 0.25% (v/v) Triton X-100, 1x Roche cOmpleteTM, EDTA-free Protease Inhibitor Cocktail) for 10 minutes at 4 °C with rocking, pelleted at 1350 x g for 5 minutes at 4°C, then Lysis Buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Roche cOmpleteTM, EDTA-free Protease Inhibitor Cocktail) for 10 minutes at room temperature with rocking, pelleted again at 1350 x g for 5 minutes at 4 °C, then resuspended in Lysis Buffer 3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) Na-Deoxycholate, 0.5% (v/v) N-lauroylsarcosine, 1x Roche cOmpleteTM, EDTA-free Protease Inhibitor Cocktail) and transferred to Covaris miniTUBEs. Cell lysates were sonicated using a Covaris E220 focused-ultrasonicator with the following settings to shear DNA to a size of 300-500 bp suitable for high-throughput sequencing: Fill Level 10, Duty Cycle 10, PIP 175, Cycles/Burst 200 for 80 seconds. 1/10 volume of 10% (v/v) Triton X-100 was then added to the sonicated lysate and cellular debris was pelleted at 20,000 x g for 10 minutes at 4 °C. Immunoprecipitation was then performed overnight at 4 °C with rotation using ProteinG Dynabeads (50 μL per IP) pre-coupled with antibody (5 μL per IP) in 0.5% BSA/PBS for >5 h. Antibodies used were anti-MORC2 Rabbit pAb (Invitrogen #PA5-51172), H3K9me3 Rabbit pAb (Abcam #8898), Rabbit IgG pAb (Abcam #ab46540). Following immunoprecipitation, beads were collected using a magnet and washed 5 times with Wash Buffer (50 mM HEPES-KOH pKa 7.55, 500 mM LiCl, 1 mM EDTA, 1% (v/v) IGEPAL CA-630, 0.7% (w/v) Na-Deoxycholate, then 1 time with Tris-EDTA-NaCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl). DNA was then eluted from beads by incubation in Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% (w/v) SDS) at 65 °C for 30 minutes with shaking. Bead-free supernatant was reverse crosslinked by incubation at 65 °C for 6-15 h, diluted with equal volume of TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA), then digested sequentially with RNaseA (0.2 mg/mL) at 37 °C for 2h and Proteinase K (0.2 μg/mL) at 55 °C for 2 h. Final immunoprecipitated DNA was then purified using Zymo ChIP DNA Clean & Concentrator kit and prepared for sequencing using Illumina Truseq 1⁄2 reaction library preparation kit. Libraries were sequenced on an Illumina NextSeq 2000 to produce 65bp paired-end reads.