GSM7662627: hrde-2(qe20) - IgG; Caenorhabditis elegans; OTHER
Worms were washed off plates and lightly crosslinked in 0.1% formaldehyde (ThermoFisher 28908) for 2min, then quenched in a 1 M Tris (pH 7.5) wash. Next, worms were washed in M9 media and pre-chilled Nuclei Purification Buffer+ (50 mM HEPES pH 7.5, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.1% Tween-20, 0.5 mM PMSF, 0.2 mM DTT, 0.5 mM spermidine, 0.25 mM spermine, cOmplete protease inhibitor cocktail (Millipore Sigma 11873580001)). Worms were resuspended in 7 ml pre-chilled Nuclei Purification Buffer+ and dounced on ice in a Wheaton dura-grind stainless steel dounce tissue grinder (VWR 62400-675) for 12 strokes. After every 5 strokes, the samples were incubated on ice for 5 min. After grinding, samples were vortexed for 30 s then incubated on ice for 5 min to release the germline nuclei twice. The nuclei were passed through six 40 μm cell strainers (Fisherbrand 22-363-547) then passed through four 20 μm cell strainers (Pluriselect NC1004201) to remove worm debris. Isolated nuclei were pelleted at 4100 rpm at 4°C for 4 min and resuspended in Nuclei Purification Buffer+, transferred to a nonstick 2 ml tube (Ambion AM12475) and an aliquot was DAPI-stained and counted to calculate the number of nuclei extracted using Hausser Scientific hemacytometer (VWR 15170-263). The remainder of the nuclei were pelleted, the supernatant was removed, and the nuclei were resuspended in NE1 buffer (1 ml 1M HEPES-KOH pH 7.9, 500 μL 1M KCl, 25 μL 1 M spermidine, 500 µL 10% Triton X-100, and 10 ml glycerol in 38 ml dH2O, 1 Roche Complete Protease Inhibitor EDTA-Free (Millipore Sigma 11873580001)), followed by slow freezing and storage at −80°C. CUT&Tag-seq was performed following the protocol (Kaya-Okur et al. 2019). 50,000 isolated C. elegans germline nuclei were thawed at 37°C and resuspended in 50 ml NE1 buffer, ConA beads were washed in binding buffer (200 μL 1M HEPES pH 7.9, 100 μL 1M KCl, 10 μL 1M CaCl2 and 10 μL 1M MnCl2, in 10 mL final volume of dH2O). 10ul ConA beads were added into 50,000 nuclei for each sample. anti-H3K27me3 antibody (Cell Signaling 9733T) was used at 1:50 for positive control, no primary antibody was used for negative control. anti-H3K9me3 antibody (Abcam ab8898) were used at 1:100 for the experiment. Samples were incubated at 4°C overnight. Anti-Rabbit IgG (Antibodies-online ABIN101961) antibody was added 1:100 to samples as the secondary antibody and incubated at room temperature for 30 mins. 2ul pA-Tn5 (EpiCypher 15-1017) was added to each sample and tagmentation was performed. DNA was isolated using Phenol:Chloroform:Isoamyl Alcohol (ThermoFisher 15593031) and chloroform. After PCR amplification using NEBNext High-Fidelity 2 × PCR Master Mix (VWR 102500-096) and post-PCR cleanup using SPRI beads (Beckman Coulter A63880).