GSM3965819: H3K79me2_DMSO; Mus musculus; ChIP-Seq
For Thio-ChIP-Sequencing, cells were scraped and collected by centrifugation at 1350 rpm for 5 min at 4o C, resuspended in 6 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, Halt phosphatase and protease inhibitors) and sonicated for 10 cycles 30 s ON and 30 s OFF, using Diagenode Bioruptor Pico. The lysates were then cleared by centrifugation at 16000 x g for 5 min, the pH was adjusted to 6.0 with formic acid, and p-nitrobenzyl mesylate was added to 1.5 mM for 30 min. Two percent of the labeled and control alkylated lysates volume were saved as input controls and the rest was incubated with 10 μl of anti-thiophosphate ester antibody overnight at 4o C. Fifty μl of protein G Dynabeads (Invitrogen) were then added and the reactions incubated for another 5 h. Beads were then washed twice with RIPA buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% sodium deoxycholate), twice with RIPA buffer with 0.3 M NaCl, twice with LiCl buffer (250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and two times with TE buffer (1 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.1% Triton X-100). The beads and the previously saved input controls were then rotated with 300 μl elution buffer (1% SDS, 0.1 M NaHCO3) overnight, 65o C. Next day, the beads were discarded and DNA recovered by adding 1.5 µl RNase A (10 mg/ml) and incubated for 30 min at 37o C. Next, 2.4 μl EDTA (0.5 M), 4.8 μl Tris-HCl (1 M), 2 μl glycogen (20 mg/ml) and 3 μl proteinase K (20 mg/ml) were added, followed by 2 h incubation at 62o C. DNA was then extracted using phenol/chloroform, washed with 75% ethanol, air dried and reconstituted in 24 μl TE buffer. For H3K79me2 ChIP-sequencing, cell pellets were thawed into Nuclei EZ lysis buffer (Sigma) with protease inhibitor cocktail (Roche), incubated for 30 min on ice, centrifuged as above, and the nuclei resuspended in 3 ml of sonication buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, protease inhibitors). Nuclei were then sonicated 10x 30 s ON (output level 3, 24W) / 60 s OFF cycles with probe sonicator, on ice. Sonicated nuclear extracts were then cleared by centrifugation at 16000 x g, 15 min, 4o C. Twenty five microliters of the nuclear extracts were saved as input material. Chromatin was immunoprecipitated from nuclear extracts with anti-histone H3 Lys79me2 antibody beads (obtained by binding ab3594 antibody to protein G Dynabeads, Novex), overnight at 4o C. The beads were collected using magnetic separator, washed, and chromatin was eluted for 30 min at 65o C with 200 μl of buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 1% SDS. Crosslinking of ChIP and input samples was reversed for 16 h at 65o C. Samples were treated with RNase A at 0.5 mg/ml for 2 h at 37o C, supplemented with 5 mM CaCl2 and 0.3 mg/ml Proteinase K and digested for 30 min at 55o C. DNA was next purified with phenol-chloroform-isoamyl alcohol mix (Qiagen) and 2 ml MaXtract gel tubes (Qiagen). After 10 min centrifugation at 16000 x g, DNA was precipitated by adding 16 μl of 5 M NaCl, 1 μl of 20 mg/ml glycogen (Fermentas) and 900 μl ethanol, centrifuged at 16000 x g, 4o C, and dissolved in 25 μl of TE buffer. For RNA-seq, total RNA was purified using RNeasy Mini Kit (Qiagen). DNA libraries were prepared according to Illumina's standard protocols. RNA libraries were prepared using Roche Kapa mRNAseq Hyper prep according to manufacturer's protocol.