title: GSM5104089: lin-53 RNAi input DNA-rep1 (ChIP-seq); Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: Mixture stage worms on RNAi NGM plates were harvested and washed three times with M9 buffer. 0.4 ml of packed worms were obtained and frozen into small balls by liquid nitrogen for each ChIP replicate. We quickly grounded the little worm balls into a frozen powder in liquid nitrogen. Worm powder was crosslinked in 4 ml crosslinking buffer (1.1% formaldehyde in PBS with protease and phosphatase inhibitors) with constant rotation for 15 min at room temperature. Fixed worm samples were quenched by incubation with 0.125 M glycine for 5 min and then washed in cold PBS-PIC (PBS buffer with protease inhibitor cocktail) three times. Tissue pellet resuspended in PBS-PIC was homogenized by 2-3 strokes in a Dounce homogenizer. The cell suspension was centrifuged at 2,000 g for 5 min at 4°C, and the supernatant was removed. We performed nuclei preparation, chromatin digestion, chromatin immunoprecipitation, and DNA Purification using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling Technology, #9003). Samples were sonicated using a Qsonica sonicator (Qsonica Q800R) at 70% amplitude for 5 cycles of 30 sec on and 30 sec off with 4 repeats. The chromatin was digested using 6 μl of micrococcal nuclease (MNase) (CST catalog number 10011S) in 300 μl of buffer B (CST catalog number 7007) containing 0.5 mM DTT for 8 min at 37 °C. The quality of chromatin digestion was analyzed by Agilent 2100 bioanalyzer system. Before IP, the chromatin sample concentration was assessed by the BCA Protein Assay Kit (Tiangen, cat no. PA115-02). 1 μg H3K27ac antibody (Abcam ab4729) or 2 μg IgG was used per IP with 0.5-1 mg input chromatin protein. According to the manufacturer, we used 200 ng purified DNA samples to construct the ChIP–seq library using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme ND607).