title: GSM8336893: H4K20me3.48h.rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Briefly, cells were crosslinked with 1% formaldehyde at room temperature for 10 mins, and quenched with 0.125 mM Glycine at room temperature for 5 minutes. The cells were then washed 3 times with ice cold DPBS and incubated with seven ml swelling buffer (25mM HEPES-KCl, pH7.2, 1.5 mM MgCl2, 20mM KCl, 0.1% NP-40) per 15cm dish incubated for 10 minutes at 4 °C. The cells were scrapped off the plates, collected and pelleted in 15 ml falcon tubes and immediately snap frozen in the liquid nitrogen, and stored in -80 °C freezer. On the day of sonication, the crosslinked cells were incubated with lysis buffer 1 (50 mM HEPES-KOH, pH7.5, 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM DTT, and 1x complete protease inhibitors cocktail) and lysis buffer 2 (10 mM Tis-HCl, pH8, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 1x complete protease inhibitors cocktail) for 5 minutes each at 4 °C. The nuclei were then resuspended in the lysis buffers 3 (10 mM Tis-HCl, pH8, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1x complete protease inhibitors cocktail) for 10 minutes on ice, followed by centrifugation at 1350g for 5 minutes. The nuclei pellets were then resuspended in lysis buffer 3 and sonicated with Covaris with (Incident peak power: 140; Duty Cycle: 5; Burst/Cycle: 200) for 15 minutes. The sonicated chromatin was centrifuging at 20,000g for 20 minutes and the soluble chromatin were collected, and 20 μl chromatin was de-crosslinked in 200 μl elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) at 65 °C over night and the rest of the chromatin was aliquoted, snap frozen in liquid nitrogen and stored in -80 °C freezer. The de-crosslinked chromatin was treated with proteinase K and RNase, followed by ethanol precipitation. For H4K20me3 ChIP-seq, 24 μg of chromatin was combined with 1.5 μg of Drosophila spike-in chromatin, and incubated with 20 μl of Protein A/G dynabeads, 3 μg of H4K20me3 (Millipore, 07-463) and 0.5 μg of drosophila spike-in antibody on a rotating wheel at 4 °C overnight.. The chromatin-antibody-dynabeads slurry was then washed 5 times with The RIPA buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCl, 1mM EDTA, 1% NP40, 0.7% Na-Deoxycholate, 1x complete protease inhibitors cocktail) and once with TE (10 mM Tris-HCl, pH 8.0, 1mM EDTA). The chromatin was eluted with elution buffer by incubating at 65 C for 20 minutes and de-crosslinked at 65 °C over night, followed by proteinase and RNase treatment and ethanol precipitation. The DNA concentration was quantified with Qubit HS DNA kit and stored in -20 °C. ChIP-seq DNA libraries was prepared with NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645) following the manufacturer's instructions. Briefly, 10 ng of ChIP DNA was used for each library and amplified with 11 PCR cycles and the library DNA was purified with Ampure XP beads and the distribution of the DNA fragments of the libraries was analyzed using High Sensitivity DNA ScreenTape (Agilent, 5067-5584) on Agilent Tapestation 4150.