GSM8818909: MNase_CT_2; Homo sapiens; MNase-Seq
Cells for Mnase-seq were crosslinked with 1% paraformaldehyde for 10 min and then quenched in 125 mM glycine for 5 min at room temperature with rotation. After washed with cold PBS, cells were lysed in lysis buffer (10 mM Tris_x0002_HCl, pH 7.5, 10 mM NaCl, and 0.5% NP-40) on ice for 10 min. The nuclei were spun down and then digested in 500 µL MNase digestion buffer (20 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, and 1 mM CaCl2) with 1000 units of MNase (NEB, Cat. #M0247S) by incubating at 37 °C for 20 min. The reaction was stopped by adding 2X STOP buffer (100 mM Tris-HCl pH 8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, and 0.2% sodium deoxycholate). After centrifuged at 15,000 × g at 4 °C for 10 mins, the supernatant was collected, adding the same amounts of Drosophila S2 cell chromatin as spike-in for normalization.  The sample was then treated with proteinase K and RNase A, and purified with the Min-Elute PCR purification kit (Qiagen, Cat. #28006). Sample extracted was subjected to library preparation with VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Cat. #ND607-1) following the manufacturer's instructions.