Syto 13 green fluorescent nucleic acid stain

To obtain diploid GM12878 for single-cell studies, cultured GM12878 were washed twice in cold PBS, fixed by resuspending them in ice cold 50% ethanol, and incubated on ice for 20–30 min. Fixed cells were pelleted, resuspended at 1 × 10⁷ cells/ml in cold PBS, and incubated on ice for 45 min with 1ug/ml RNase A (Qiagen) and 500 nM SYTO™ 13 Green Fluorescent Nucleic Acid Stain (Thermo Fisher Scientific, Waltham, MA, USA). Diploid cells were collected in the G1 phase of the cell cycle using a FACS Synergy flow cytometry (iCyt Mission Technology, Champaign, IL, USA). Single cells were isolated from the diploid population using a Cell Microsystems CellRaft and Cell Microsystems CellRaft apparatus according to manufacturer's instructions. WGA was done using an Illustra GenomiPhi V2 DNA Amplification Kit (GE Biosciences) with a modified protocol. Briefly, single cells on raft were incubated for 30 min on ice in 1 ul of 20 mM KOH and 50 mM DTT then frozen at –20°C or –80°C for a minimum of 30 min and maximum of 1 week. After freezing, the cell lysate was incubated at 65°C for 10 min and then on ice for 2 min. After cooling, 9ul of sample buffer was added to lysate and incubated for 10 min on ice. Then, a mix of 9 ul of reaction buffer and 1ul of enzyme was added and incubated for 2 h at 30°C and 10 min at 75°C. Amplified DNA was purified by ethanol precipitation.