Microspin g 25 spin column

Standard 5′ radiolabeling was performed with T4 polynucleotide kinase (New England Biolabs) at 0.2 U/μL (manufacturer’s units), 1X T4 PNK buffer (New England Biolabs), 400 nM DNA oligonucleotide, and 200 nM [γ−³²P]-ATP (PerkinElmer) for 30 min at 37°C, followed by a 20-min heat-killing incubation at 65°C. Radiolabeled oligos were then buffer exchanged into water using a Microspin G-25 spin column (GE Healthcare). For 5′ radiolabeling of sgRNAs, the 5′ triphosphate was first removed by treatment with Quick CIP (New England BioLabs, manufacturer’s instructions). The reaction was then supplemented with 5 mM DTT and the same concentrations of T4 polynucleotide kinase (New England BioLabs) and [γ−³²P]-ATP (PerkinElmer) used for DNA radiolabeling, and the remainder of the protocol was completed as for DNA.