GSM7900756: FG_Ctrl_Low_MNase_CloneA6; Homo sapiens; MNase-Seq
Dissociated foregut cells were washed with Wash Buffer (20 mM HEPES-NaOH [pH. 7.5], 150 mM NaCl, 0.5 M Spermidine and 1x Complete Protease Inhibitor EDTA free), centrifuged at 600 xg for 3 min, and the supernatant was removed (twice). The cell pellet was resuspended with Digitonin Buffer (0.02% Digitonin in wash buffer). Cell suspension was divided into 1.0x106 cells per reaction, added 13 U/mL (low) or 160 U/mL MNase (LS004798, Worthington), and incubated for exactly 5 min at 37°C while shaking at 300 rpm in ThermoMixer F1.5 (Eppendorf). MNase reaction was quenched by adding equal volumes of 2x Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 0.05 mg/ml RNaseA, and 0.05 mg/ml Glycogen). For RNase reaction, samples were incubated at 37°C for 30 min. Then, samples were treated with 0.5% SDS and 0.2 mg/mL Proteinase K for 2 hours at 37°C. Finally, DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The DNA concentration was measured by nanodrop (ThermoFisher Scientific). Mono- and sub-nucleosomal DNA fragments were selected and purified by MagBind Magnetic Beads (M1378-01, Omega). The purified DNA concentration was measured by Quantus fluorometer (Promega). MNase-seq libraries were prepared by using NEBNext Ultra II DNA Library Prep Kit for Illumina with dual index primer system. 10 ng of mono-nucleosomal DNA fragments were used as starting material.  Libraries were amplified by using 5 cycles on thrmocycler. Amplified libraries were size selected at 200-600 bp by using MagBind beads from Omega. Libraries were validated by using Agilent High Sensitivity DNA Kit.