title: GSM8526773: control-IP ChIP sequencing rep. 1; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: ChIP experiments were conducted as previously described (Guang, et al. 2010; Weng, et al. 2019) with minor modifications. Briefly, 60,000 worms were crosslinked in 2% formaldehyde in M9 buffer for 30 minutes at room temperature with constant agitation. Crosslinking was quenched by adding 125 mM glycine in M9 for 5 minutes at room temperature. After washing with M9 and water, the worm pellets were resuspended in FA buffer (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl) supplemented with a protease inhibitor (Thermo). The worm pellets were then homogenized using a mixture grinder at 6 m/s for 45 seconds, repeated three times. Next, crosslinked chromatin was sonicated for 30 cycles of 30 seconds ON and 30 seconds OFF at HIGH output. After sonication, worm lysates were centrifuged, and the supernatants were immunoprecipitated with 6 μg of anti-Flag antibody. The antibody-protein-DNA complex was pulled down using Dynabeads Protein G (Invitrogen). After washing sequentially with FA buffer, FA buffer containing 500 mM NaCl, and TE buffer (pH 8.0), reverse crosslinking was carried out by adding 1x TE/1% SDS/250 mM NaCl and incubating at 65°C for 4 hours. RNA and proteins were digested by treating the samples with 20 μg of DNase-free RNase A (Invitrogen) at 37°C, followed by incubation with 200 μg of proteinase K at 60°C for 1 hour. DNA was extracted using phenol-chloroform and dissolved in Tris buffer. Sequencing libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (NEB, ctalog: E7103), following manufacturer's instructions.