GSM4932460: soma-control-HS-HSF1_ChIP_replicate2; Caenorhabditis elegans; ChIP-Seq
Animals were collected from NGM plates, washed with M9 and crosslinked with 2% formaldehyde in PBS at room temperature for 15 min. Crosslinking was quenched by incubation with 0.1M Tris (pH 7.5) for 5 min. Worms were then washed three times in M9 and once in cold FA buffer (50 mM HEPES/KOH pH7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxyholate, 150 mM NaCl with Roche Complete protease inhibitors). Worms were then resuspend in 600 ul of FA buffer and lysed by douncing in a Kontes 2 ml glass dounce, and sonicated in Bioruptor to yield 200 bp–600 bp size DNA fragments. Immunoprecipitation (IP) was set up using 0.5 ug of chromatin, and either 5 ul of anti-full length GFP antibody (Clontech, living colors), or 3 ug of anti-RNA Pol II antibody (8WG16, BioLegend) in FA buffer (1 ml total volume), and incubated at 4 ºC overnight on a rotating wheel. The next day, 30 ul of protein-G Dynabeads were added to each IP. ChIP-seq libraries were prepared using Accel-NGS 2S kit as per manufacturer's instructions.