Terminal transferase

For the DNA binding experiments, single-stranded DNA oligonucleotide (93 nt long, X12-3HJ3)^{69 (link)} was labeled at the 5′ terminus with [γ-³²P] ATP and T4 polynucleotide kinase (New England Biolabs), according to standard protocols. Unincorporated nucleotides were removed using Micro Bio-Spin™ P-30 Gel Columns (Bio-Rad). Plasmid length DNA binding experiments were performed with unlabeled M13mp18 single-stranded DNA (New England Biolabs).
For helicase assays, oligonucleotide containing a 37 nt region complementary to the M13mp18(+) strand (nucleotides 6289–6326) and a 40 nt tail at the 5′ end was annealed to M13mp18 single-stranded DNA to prepare the substrate^{38 (link)}. The oligonucleotide was labeled at the 3′ terminus [α-³²P] dCTP (Perkin Elmer) and terminal transferase (New England Biolabs) before annealing according to the standard procedures.

To inactivate the 3’-hydroxyl terminus of DNA molecules, a single ddGMP was added to the ends of 2 μg of amplified cDNA in a 50 μL mixture containing 0.1 mM ddGTP (Roche, 03732738001), 0.25 mM CoCl₂ (NEB, M0315), 20 U of Terminal Transferase (NEB, M0315) and 1× TdT buffer (NEB, M0315). The reaction was performed at 37 °C for 2 h and stopped by heating for 20 min at 75 °C. Thereafter, the DNA sample was purified using AMPure XP beads and eluted in 24 μL of nuclease-free water. Three microliters of Quick CIP enzyme (NEB, M0525) and 3 μL of 10× Cutsmart buffer (NEB, B7204) were added to the eluate, and the mixture was incubated at 37 °C for 30 min for dephosphorylation. After inactivation of the phosphatase at 80 °C for 5 min, the DNA molecules were cut with 10 pmol of Cas9-gRNA complex (2.5 pmol each) at 37 °C for 15 min. For the removal of Cas9 protein from cleavage sites, the sample was digested with 1.5 μL of protease (Qiagen, 19155) for 10 min at 56 °C, followed by 70 °C for 15 min to inactivate protease. dA-tailing was carried out at 72 °C for 5 min with 1 μL of 10 mM dATP (NEB, N0440) and 5 U of Taq DNA polymerase (Vazyme, P101-d1-AC). The product was used directly for adapter ligation through SQK-LSK110 (ONT) or barcoding through NBD114 (ONT).