GSM8389104: Input of condition hpl2, ID:CITNR; Caenorhabditis elegans; ChIP-Seq
ChIP was conducted with slight adjustments following the method described by Ercan et al. (2007). Post-crosslinking, C. elegans embryos underwent a wash with 100 mM Tris-HCl, pH 7.5, followed by two washes with FA buffer (50 mM HEPES-KOH, pH 7.5; 1 mM EDTA; 1% (v/v) Triton X-100; 0.1% (w/v) sodium deoxycholate; 150 mM NaCl; 1x protease inhibitor (cOmplete™, Sigma, Germany)). Nuclei were sheared as for Hi-C and then centrifuged at 13,000 xg for 15 minutes at 4°C to gather extracts. Immunoprecipitations were arranged using extracts equivalent to 3 mg along with 3–5 μg of antibodies in a total volume of 500 μL FA buffer. Prior to antibody addition, 10% of the reactions were kept aside as input. After 16 hours of incubation at 65°C with agitation, complexes were recovered by adding 20 μL of Protein A/G beads (Pierce™ Protein A/G Magnetic Beads, USA) and washed twice with FA buffer supplemented to 1 M NaCl, followed by two washes with FA buffer supplemented to 500 mM NaCl. Beads were treated with 1% (w/v) SDS in TE containing 250 mM NaCl at 65°C for 30 minutes to elute the complexes. Samples and inputs were treated with 2 μL of 10 mg/mL Proteinase K for 1 hour at 55°C. Decrosslinking was carried out at 65°C overnight in a thermomixer. DNA purification was executed with a PCR clean-up kit (Macherey-Nagel™, Germany) and quantified using Qubit Fluorometric Quantification. Library preparation followed the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, USA) protocol. Libraries were sequenced on Novaseq 6000 PE150 S4 flow cells. Various antibodies were employed for ChIP experiments: anti-HPL-2 (Novus Biologicals 38630002), anti-LIN-61 (Novus Biologicals 29590002), anti-LEM-2 (Novus Biologicals 48540002), anti-H3K9me2 (Abcam ab1220), anti-H3K9me3 (VWR 309M3-B), anti-H3K4me1 (Abcam ab8895), anti-H3K4me3 (Active Motif 39159), anti-H3K27me3 (Abcam ab6002), and anti-H3K27ac (Active Motif Ab39133). All ChIP-seq experiments were conducted as biological independent triplicates. Immunoprecipitation of C. elegans nuclear extracts utilized anti-LIN-61 (Novus Biologicals 29590002), anti-HPL-2 (Novus Biologicals 38630002), and anti-T7 (Abcam ab9138) antibodies. The antibodies were crosslinked to dynabeads M-280 sheep anti-rabbit IgG (Pierce, USA) overnight at 4°C on a nutator, following the manufacturer's instructions. Unbound antibodies were removed by washing the beads with 1xPBS three times. Beads were then resuspended in 1 mL 0.2 M triethanolamine, pH 8.2, 20 mM DMP at room temperature for 60 minutes. Cross-linking was halted by adding 1 mL 50 mM Tris-HCL, pH 7.5, at room temperature for 60 minutes. After three washes with 1xPBS, 5 mg of total protein C. elegans extracts in 500 μL were added to the beads and incubated for 30 minutes at room temperature. Subsequently, beads underwent six washes with buffer D (each wash involving incubation with 1 mL buffer D for 5 minutes followed by centrifugation at 2000 xg for 1 minute at room temperature). Bound proteins were released from the beads in SDS-PAGE sample buffer by incubating at 95°C for 10 minutes. The proteins were resolved on SDS-PAGE and subjected to analysis by western blotting following standard procedures.