GSM5494264: LIN14L2IPrep4; Caenorhabditis elegans; ChIP-Seq
Worms were fixed with 2% formaldehyde for 15 minutes at room temperature (RT). ChIP assay was performed as previously described with the following modifications (Li et al., 2020). After fixation, worms were resuspended in FA buffer supplemented with protease inhibitors (150 mM NaCl, 10 μl 0.1 M PMSF, 100 μl 10% SDS, 500 μl 20% N-Lauroyl sarcosine sodium, 1 tablets of cOmplete ULTRA Protease Inhibitor Cocktail in 10 ml FA buffer). The sample was sonicated using a Covaris S220 at the following settings: 200 W Peak Incident Power, 20% Duty Factor, 200 Cycles per Burst for 6 min. Samples were transferred to centrifuge tubes and spun at the highest speed for 15 min. The supernatant was transferred to a new tube, and 5% of the material was saved as input. The remainder was incubated with 25 μl GFP-Trap Magnetic Beads (Chromotek gtd) at 4°C overnight. Wild-type (N2) worms do not carry the GFP tag and serve as negative control. The next day, the beads were washed at 4°C twice with 150 mM NaCl FA buffer (5 min each), once with 1M NaCl FA buffer (5 min), twice with 500 mM NaCl FA buffer (10 min each), once with TEL buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) for 10 min, and twice with TE buffer (5 min each). The immunocomplex was then eluted in 200 μl elution buffer (1% SDS in TE with 250 mM NaCl) by incubating at 65°C for 20 min. The input and ChIP samples were then treated with 1μl of 20 mg/ml proteinase K, incubated at 55°C for 2 hr, and then 65°C overnight to reverse cross-link. The immunoprecipitated DNA was purified with Ampure XP beads (A63881) according to manufacturer's instructions. ChIP-seq libraries were prepped using the Ovation UltralowSystem V2 kit (Tecan) according to manufacturer's protocol.