title: GSM5172396: input_wt_H3K9me2_20°C_2; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: ChIP experiments were performed as previously described (Zeller et al., 2016). In brief: Embryos were harvested from synchronized animals in three replicates. 40 μg of chromatin was incubated overnight with primary antibody (H3K27ac (Abcam, ab177178) and H3K9me2 (MBL, #MABI0317) ) coupled to Dynabeads Sheep Anti-Rabbit IgG (Invitrogen), in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl)) containing 1% SDS. Antibody bound chromatin was washed 3 × 5 min FA buffer; 5 min FA buffer with 1 M NaCl; 10 min FA buffer with 500 mM NaCl; 5 min with TEL buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0) and 2 × 5 min with TE. Complexes were eluted in 1% SDS in TE with 250 mM NaCl at 65°C for 15 min. Samples and inputs were treated with 20 μg of RNAse A for 30 min at 37°C and 20 μg of proteinase K for 1 hr at 55°C. Crosslinks were reversed by overnight incubation at 65°C. DNA was subsequently purified using Zymo DNA purification columns (Zymo Research). Libraries were prepared from chromatin IP and input samples using the NEBNext ultra DNA library prep kit for Illumina (NEB # 7370) and the NEBNext Multiplex Oligos for Illumina (NEB # E7335), according to the manufacturers recommendations. No size selection was performed during sample preparation and the libraries were indexed and amplified using 12 PCR cycles, using the recommended conditions. After a final cleanup with Agencourt AmPure XP beads (Beckman # A63881), the library size distribution and concentrations were determined using a BioAnalyzer 2100 (Agilent technologies) and Qubit (Invitrogen) instrument, respectively. The final pools were prepared by mixing equimolar amounts of all individual indexed libraries and then sequenced on a HiSeq 2500 (Illumina) in Rapid mode (Paired-End 50).