GSM8374999: Isolated germ nuclei high salt YL701 ETOH control H3  rep 1; Caenorhabditis elegans; ChIP-Seq
For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each For histone modification ChIP-seq, isolated germ cell nuclei were sonicated in a Misonix S-4000 water bath sonicator, and immunoprecipated overnight using 5 µg of anti-H3K27me3, anti-H3K36me3, or anti-H3K9me3 antibody. For each IP, 90ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. The wash steps were performed as in previously described (Han et al 2019). For Native ChIP-seq, 12 million nuclei were incubated with Lysis Buffer 1 (1 mM Tris–HCl pH7.5, 1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 0.2% NP-40, 0.01% digitonin) for 10min. Then 10U of Mnase was added and digested at 37°C for 4 min and 30 sec. The reaction was stopped with 4 μL of 0.5M EDTA and spun for 17000g for 10 min at 4°C. Supernatant was collected and nuclei pellet was incubated with lysis buffer 2 (1 mM Tris–HCl pH7.5,1 mM PMSF, 1 mM DTT, 0.1% Tween 20, 1% Triton X-100 + 1x Complete Protease Inhibitor Cocktail) for 2 hours. Supernatants were combined and chromatin was pre-cleared with blocked beads. Chromatin was immunoprecipated overnight with 5 µg of anti-H3 antibody. For each IP, 10ng of Drosophila spike-in chromatin and 0.5 μg of Drosophila-specific H2Av antibody was added. After overnight incubation, , 40μL of pre-blocked beads were added and incubated 4°C for 2 hours. For the high salt wash, the ChIP samples with beads were washed 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-500mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 500mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). For the low salt wash, the ChIP samples were washed 2 times with FA-75mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 75mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)) and 2 times with FA-150mM NaCl (50mM HEPES pH 7.5, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% Na deoxycholate, 150mM NaCl and 1x cOmplete proteinase inhibitor cocktail EDTA-free (Roche)). DNA was eluted twice with elution buffer (100mM NaHCO3, 0.2% SDS, 5 mM DTT, 1x TE) at 65°C for 15 min. Samples were treated with RNAse, and incubated with proteinase K overnight at 55°C. DNA was purified on Zymo ChIP DNA Clean & Concentrator kit (Zymo Research #D5205) and used for ChIP library preparation. Samples were sequenced on an Illumina NovaSeq using 100bp paired-end sequencing.