Nascent strand DNA sequencing (NS-seq) for re-replicating cells

HCT116 cells were treated with 250 nM of MLN4924 for the indicated time in order to have most or all cells in re-replicating cycle. Cells incubated without MLN4924 were used as control. Genomic DNA was purified by phenol/chloroform and ethanol precipitation and used to isolate nascent strands. To isolate nascent strands, DNA was denatured by boiling for 10 min, immediately cooled on ice, and fractionated on a neutral 5–30% sucrose gradient. Gradients were centrifuged at 50,000 × g for 18 h with an SW40 swing bucket rotor. Fragments, 0.5–2 kb, (containing nascent strand DNA and broken genomic DNA) were collected and treated with λ exonuclease to remove non-RNA-primed broken genomic DNA. The remaining single-stranded nascent strand DNA was converted to double-strand DNA using the BioPrime DNA Labeling System (ThermoFisher, 18094011). Double-stranded nascent DNA (1 μg) was sequenced using the Illumina genome analyzer II (Solexa). Sheared genomic DNA was also sequenced to be used for peak calling.

HCT116 cells were treated with MLN4924 for 24 h. BrdU was added to cells 8 h after MLN4924 treatment for a total of 16 h of BrdU incorporation, which was less than one doubling time. DNA was purified from these cells and sonicated to 3–10 kb. Re-replicated DNA (in which both DNA strands had undergone BrdU incorporation) was isolated using a BrdU-CsCl gradient. Re-replicated DNA and some DNA that had not undergone re-replication (only one strand having incorporated BrdU) were collected for NS-seq as described below.