Nascent Strand Sequencing

NS-Seq sequences nascent DNA strands to locate DNA replication origins in the genome. NS-Seq uses _-exo to digest parental DNA effectively while leaving the RNA primer_protected nascent strands intact. However, _-exo inefficiently digests G-quadruplex structures (G4) and GC-rich motifs; this bias can be normalized by using _-exo_digested DNA from nonreplicating cells as a control (Foulk et al., 2015).

In this method, gDNA is enriched, made single-stranded, and 5ê-phosphorylated using T4 PNK. Next, the DNA is digested with _-exo and purified. The resultant single-stranded nascent strands are converted to double-stranded DNA using random hexamers and fragmented to 100_600 bp. DNA libraries are prepared, using standard kits, and sequenced.

• Locates DNA replication origins by sequencing RNA primer_protected nascent DNA strands
• _-exo_digested DNA from nonreplicating cells can be used as a control to normalize for biases in _-exo digestion
• Replacing K+ with Na+ during _-exo digestion reduces digestion inefficiency in G4 regions

• _-exo does not efficiently digest G4 structures in plasmid and halts upon GC-rich motifs
• Purified samples can be contaminated with GC-rich and G4-protected DNA

Nascent Strand Sequencing

NS-Seq sequences nascent DNA strands to locate DNA replication origins in the genome. NS-Seq uses _-exo to digest parental DNA effectively while leaving the RNA primer_protected nascent strands intact. However, _-exo inefficiently digests G-quadruplex structures (G4) and GC-rich motifs; this bias can be normalized by using _-exo_digested DNA from nonreplicating cells as a control (Foulk et al., 2015).

In this method, gDNA is enriched, made single-stranded, and 5ê-phosphorylated using T4 PNK. Next, the DNA is digested with _-exo and purified. The resultant single-stranded nascent strands are converted to double-stranded DNA using random hexamers and fragmented to 100_600 bp. DNA libraries are prepared, using standard kits, and sequenced.

• Locates DNA replication origins by sequencing RNA primer_protected nascent DNA strands
• _-exo_digested DNA from nonreplicating cells can be used as a control to normalize for biases in _-exo digestion
• Replacing K+ with Na+ during _-exo digestion reduces digestion inefficiency in G4 regions

• _-exo does not efficiently digest G4 structures in plasmid and halts upon GC-rich motifs
• Purified samples can be contaminated with GC-rich and G4-protected DNA