GSM6600072: H3K79me1_fl_rep2; Mus musculus; ChIP-Seq
Cells were fixed for 10 minutes with 1% formaldehyde, in solution, rotating at room temperature. Fixing was quenched with 0.14 M glycine for 5 minutes, and the cells were spun down at 300xg for 3 minutes. Cells were washed 3 times with PBS and flash frozen. 25-30 million cells were resuspended in 1 mL lysis buffer (1% SDS, 50 mM Tris-HCl pH 8, 20 mM EDTA, 1x cOmplete (Roche, 4693132001) protease inhibitor), and sonicated on a Covaris S220 Focused-ultrasonicator with the following parameters: 21 cycles of 45 seconds ON (peak 170, duty factor 5, cycles/burst 200), 45 seconds OFF (rest) in 6-8°C degassed water to generate fragments of 200-400 bp. Sonicated DNA was quantified with Qubit dsDNA HS (ThermoFisher Scientific, Q32854), aliquoted, and stored at -80°C. The immunoprecipitation was set up with 7.1 ug of assay chromatin plus 1:53 (133 ng) of human 293T spike in chromatin, generated as above. Chromatin was diluted 1:10 with dilution buffer (16.7 mM Tris-HCl pH 8, 0.01% SDS, 1.1% Trition-X, 1.2 mM EDTA, and 167 mM NaCl), and immunoprecipitated with 5 ug H3K79me1 (Abcam, ab2886, Lot GR274715-4) or H3K79me2 (Active Motif, 39143, Lot 16918002) antibody for 16 hours, rotating at 4°C. 25 μL of Protein A (ThermoFisher Scientific, 10002D) and 25 μL of Protein G (ThermoFisher Scientific, 10004D) Dynabeads that were washed once in PBS, 0.02% Tween-20 and once in dilution buffer, were added. Chromatin was incubated, rotating, with beads for 2 hours at 4°C. Beads were washed twice, rotating at 4°C for 5 minutes, with 1 mL of each of the following buffers: Low salt (50 mM HEPES pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1 mM EDTA pH 8.0, 140 mM NaCl), High salt (50 mM HEPES pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1 mM EDTA pH 8.0, 500 mM NaCl), LiCl (20 mM Tris-HCl pH 8, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA pH 8.0, 250 mM LiCl), and TE (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8) using a magnetic rack. Beads were incubated with 250 μL elution buffer (50 mM Tris-HCl pH 8, 1 mM EDTA pH 8, 1% SDS) for 10 minutes at 65°C, shaking. 200 μL of TE with 0.67% SDS was added, and incubated shaking at 65°C for 10 minutes. 10 μg RNase A was added and incubated at 37°C for 30 minutes. 40 μg of Proteinase K was added, and incubated for 12-16 hours at 65°C to reverse crosslinks. DNA was cleaned with phenol-chloroform phase separation with phase lock tubes (VWR, 10847-802). DNA was precipitated with 1 volume isopropanol, 1/10th volume of 3M NaActate, and 20 μg glycogen, and washed with 70% ethanol. DNA was dissolved in 30 μL ultrapure water and concentrated to 5 μL using a SpeedVac for library preparation. The entire ChIP sample was used as starting material for library construction with Ovation Ultralow System V2 using reagents at 0.5x and adapters diluted 1/5 in water, following the manufacturer's instructions (NuGEN, 0344). The libraries were separated on an 1.5% agarose gel, cut from 200-400 bp for an additional size selection step. The gel fragment was purified using the MinElute Gel Extraction Kit (Qiagen, 28604).