Repli-seq in HeLa cells of late stages of S-phase
HeLa S3 cells were synchronised at the G1/S border by single thymidine arrest as described before and released in S phase for 2 h (early S phase), 3.5 h (Mid-S phase) and 5.5 h (late S phase). 10x107 cells were used per time-point. DNA was labelled for 20 min with 20 uM EdU and washed with PBS containing 10 uM thymidine. Cells were resuspended in ice cold 0.25% TritonX/PBS at 1.25x107 cells/mL and incubated at RT for 30 min. Samples were washed in ice cold 0.5% BSA/PBS and resuspended in click-IT reaction (10 uM Biotin-azide, 10 mM Na Ascorbate, 1 mM CuSO4), using 0.5 mL per 1x107 cells. The click-IT reaction was incubated at RT for 1 h. Samples were washed with ice cold 0.5% BSA/PBS and PBS. Cells were sonicated in lysis buffer (50mM Tris HCl pH 8.0, 1% SDS) for 24 cycles (30 sec. ON/90 sec. off) and spinned at max speed for 30 min. 20 uL of Dynabeads per sample were added and incubated over-night at 4 C. Beads were washed 4 times with B&W buffer (10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA, 0/1% Tween-20), twice with 0.05% Tween-20 in TE, once with 10 mM Tris-HCl ph 7.5 and resuspended in 100 uL EB buffer. To elute, samples were incubated in 50 ug/mL RNAse for 30 min at 37 C and 0.5% SDS and 1 mg/mL proteinase K over-night at 37 C and at 65 C at least 6 h. 100 uL of TE was added and DNA was purified following the Monarch PCR & DNA clean-up kit instructions. Library preparation was done following the instructions of the NEBNext Ultra II kit for Illumina sequencing.