Synthesis of Labeled Single-Stranded DNA

PAGE-purified oligos were purchased from IDT. ssDNA circles were prepared by annealing 5 μM phosphorylated template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG) and 4.5 μM biotinylated primer oligo (5/Biosg/TC TCC TCC TTC T) in 1× T4 ligase reaction buffer (NEB B0202S).^{31 (link),32 (link)} Oligos were heated to 75 °C for 5 min and cooled to 4 °C at a rate of −1 °C min⁻¹. Annealed circles were ligated with the addition of 1 μL of T4 DNA ligase (NEB M0202S) at room temperature for ~5 h. Ligated circles can be stored at 4 °C for up to 1 month. Low-complexity ssDNA was synthesized in 1× phi29 DNA polymerase reaction buffer (NEB M0269S), 500 μM dCTP and dTTP (NEB N0446S), 0.2 mg mL⁻¹ BSA (NEB B9000S), 10 nM annealed circles, and 100 nM phi29 DNAP (purified in-house). The solution was mixed and immediately injected into the flow cell and incubated at 30 °C for 20 min. For digoxigenin incorporation as an end label, 100 μM dTTP was included in a 5, 50, or 500 molar excess over digoxigenin-11-ddUTP (Roche). ssDNA synthesis was quenched by removing excess nucleotides and polymerase with BSA buffer (40 mM Tris-HCl pH 8.0,2 mM MgCl₂, 1 mM DTT, and 0.2 mg mL⁻¹ BSA).