GSM4498734: U2OS_M_phase_plus_Aphidicolin_negative_control_1; Homo sapiens; OTHER
Cells were lysed in the Lysis buffer (50mM Tris-HCl, pH 8.0, 1% SDS, 20mg/ml RNase A, 20mg/ml protease K) in a 37˚C water bath for over 6h. The DNA was extracted using the phenol/chloroform method and sonicated to an average size of 300-400 bp using a Bioruptor (Diagenode). DNA library was constructed with KAPA Hyper Prep Kit (KK8502). Briefly, 3ul of Enzyme Mix and 7ul of corresponding buffer were added to beads adsorbed with biotin-DNA. End repair and an A-tailing reaction were performed in a thermocycler according to the manufacturer's protocol. Adaptor stock was diluted to 300nM and adaptor ligation was accomplished at 20˚C for 0.5h. The beads were washed with a double volume of the TE buffer (10 mM Tris-HCl, pH 8, and 1 mM EDTA) at least three times to remove unligated adaptor and adaptor-dimer molecules. Each library amplification reaction contained KAPA HiFi HotStart ReadyMix and the Primer Mix (10X) and was carried out using amplification for 15 cycles. Reactions were cleaned up using AMPure XP (Beckman Coulter, A63880).