GSM5920751: LacZ_RNAi_Rep1_MNase; Drosophila melanogaster; MNase-Seq
Cells were treated with 1% formaldehyde for 2 min at room temperature, and cross- linking was quenched by adding glycine to 125 mM and placing the cells on ice. Nuclei were isolated as for PRO-seq (KWAK et al. 2013), except they were stored in MD buffer (10 mM Tris- HCl pH 8.0, 250 mM sucrose, 1 mM CaCl 2, 60 mM KCl, 15 mM NaCl, 1 mM DTT). MNase digestions were done in 140 μl MD buffer on nuclei from 1.5 x 10e7 cells with 4000 U MNase (NEB gel units) at room temperature for 30 min, resulting in around 90% mononucleosomes. Reactions were stopped (360 μl 142 mM NaHCO 3, 1.42% SDS, 350 mM NaCl, 17.5 mM EDTA, 2.8 mM EGTA), DNA was isolated, and MNase-seq libraries were prepared as previously described (WEI et al. 2012). After 4 PCR cycles, DNA in the size range of 80 to 220 bp (not including adapter sequences) was isolated and sequenced.