GSM6068863: sYW-2-RNHinput; Saccharomyces cerevisiae; OTHER
For DRIP-seq, genomic DNA was isolated using Qiagen Tip kit according to manufacturer's protocol with the exception that RNase was not added to buffer G2. Genomic DNA was first treated with S1 nuclease for 30 minutes at 50°C (as described in (Wahba et al., 2016b)). Samples were then fragmented by sonication to ~300 bp. For qPCR analysis, genomic DNA was isolated by phenol:chromoform extract followed by a brief treatment with RNaseA/T1 in 300 mM NaCl. DNA was fragmented by overnight digestion at 37°C with restriction enzymes (AccI, EcoRV, NcoI, HaeIII, BsrG1) in cutsmart buffer in the presence or absence of 20 U RNaseH. For DRIP-seq and DRIP-qPCR, following fragmentation, samples were purified by phenol:chloroform extraction then ethanol precipitated. Samples were resuspended in 1XFA buffer (1% TritonX-100, 0.1% sodium deoxycholate, 0.1% SDS, 50 mM HEPES, 150 mM NaCl, 1 mM EDTA). Antibody S9.6 (25µg) was pre-bound to 100 µL Protein A Dynabeads for 1 hour at 4°C in 1XFA buffer. 100 µg fragmented DNA was incubated with the S9.6-protein A beads for 2 hours at 4°C. Beads were washed in 5 successive washes: 1XFA, 1XFA with 0.5M NaCl, LiCl wash buffer (0.25M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl), twice with TE buffer. DNA was eluted in 300 µL elution buffer (1%SDS, 0.1M NaHCO3) for 2 hr at 65°C. Samples were then treated with 1 µL Proteinase K (20 mg/mL) for 1 hour at 42°C, extracted with phenol:chloroform, and then ethanol precipitated. The final DNA pellet was resuspended in 50 µL TE buffer. Libraries were prepared with Qiagen GeneRead Kit according to manufacturers instructions.