GSM8862624: mig-32(n4275) ChIP-seq, H2AK119ub, replicate 2; Caenorhabditis elegans; ChIP-Seq
Embryos were collected by centrifugation (1000g, 3 min.) and washed twice in phosphate-buffered saline (PBS) containing 0.01% (v/v) Triton X-100 (PBS/Tx). To crosslink, PBS/Tx was added to at least 80 times the pellet volume, and 37% formaldehyde (Sigma F8775) was added to a final concentration of 1.8%. Samples were incubated at room temperature with rotation for 25 minutes. Glycine was added to a final concentration of 125 mM followed by incubation for an additional 5 minutes. All subsequent steps were performed on ice using pre-chilled buffers and tubes. Embryos were washed twice with PBS/Tx as above and an equal volume of resuspension buffer (50 mM HEPES-KOH pH7.5, 150 mM NaCl, 1 mM EDTA, 0.01% Triton X-100, protease inhibitors (1 tablet per 5 mL, Roche 4693159001) was added to the embryo pellet. The resuspended embryos were aliquoted, snap-frozen in liquid nitrogen and stored at -80°C. Aliquots of cross-linked embryos were thawed in an ice-water bath, the volume was adjusted to 3-4 pellet volumes (PV) using resuspension buffer, and 100 uL was aliquoted to each polystyrene sonication tube (Evergreen 214-3721-010). An equal volume of resuspension buffer containing 2X detergents was added (50 mM HEPES-KOH pH7.5, 150 mM NaCl, 1 mM EDTA, 0.2% sodium deoxycholate, 0.7% sodium sarkosyl). Samples were sonicated in a Q800R3 water bath sonicator (QSonica Q800R3-110; 50% power, 20s on/20s off) for 4 minutes. Samples were gently mixed by pipetting and sonicated an additional 4 minutes. The lysate was transferred to an Eppendorf tube and an equal volume of resuspension buffer without detergents was added (50 mM HEPES-KOH pH7.5, 150 mM NaCl, 1 mM EDTA). The lysate was centrifuged (13,000g, 15 min. 4°C) and the supernatant was transferred to a new tube. Protein concentration was determined using the Bradford assay (Sigma B6916). As a spike-in control for calibration, sonicated Drosophila chromatin (Active Motif, 53083) was added at a ratio of 1:20. The supernatant was aliquoted to tubes for immunoprecipitation (IP), saving 20% of the volume of one IP tube as the 'input' control, which was stored at -20°C. Triton X-100 (10%, v/v) was added to a final concentration of 1% (v/v). This step was omitted for anti-H3K27me3 (CST 9733, C36B11), for which this detergent led to increased background. After antibody addition, samples were incubated overnight at 4°C with rotation. Protein G dynabeads (Invitrogen 10003D; 7.5 uL per IP) were pre-washed twice with resuspension buffer and added to IPs, which were then incubated as above for 2 hours. Beads were collected on a magnetic stand (Permagen MSR812) and washed as follows, with each wash incubated for 5 minutes at 4°C with rotation: 2X with FA-150 (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate), 1X with FA-1M (FA-150 with 1 M NaCl), 1X with FA-0.5M (FA-150 with 0.5 M NaCl), 1X with TE-LiCl in a new tube (10 mM Tris-Cl pH 8, 1 mM EDTA, 250 mM LiCl, 1% IGEPAL CA-630, 1% sodium deoxycholate), 2X with TE+ (10 mM Tris-Cl pH 8, 1 mM EDTA, 50 mM NaCl, 0.005% IGEPAL CA-630). Elution was performed in a new tube at 65°C for 15 minutes using 50 uL of ChIP elution buffer (10 mM Tris-Cl pH 8, 1 mM EDTA, 200 mM NaCl, 1% SDS). Elution was repeated and the supernatants were pooled. The 'Input' aliquot was thawed and brought to 100 uL with ChIP elution buffer. Input and IP samples were treated with RNase A (16.5 ug, Sigma R4642) for 1 hour at 37°C and proteinase K (40 ug, Bioline BIO-37084) for 2 hours at 55°C. Crosslinks were reversed by incubation overnight at 65°C. DNA was isolated using a QIAquick PCR purification kit (Qiagen 28104). Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645S) according to the manufacturer's instructions, using 25-50 ng of input DNA or half of ChIP DNA. Library concentration was quantified using NEBNext Library Quant Kit for Illumina (NEB E7630). Pooled libraries were sequenced using NextSeq500 in 75 bp paired-end mode.