Applied biosystems sequence scanner software v2

Two-step ORNi-PCR was performed using KOD-Plus-Ver. 2 (Toyobo) as described previously [4 (link)] and in Supplementary Protocol. Briefly, reactions were carried out with an initial denaturation at 94°C for 2 min, followed by 30–34 cycles at 98°C for 10 s and 50°C–74°C for 60–90 s. ORNi-PCR products were electrophoresed on 1−2% agarose gels and, if necessary, subjected to DNA cloning and/or DNA sequencing analysis. DNA sequencing data were analyzed using Applied Biosystems Sequence Scanner Software v2.0 (Thermo Fisher Scientific). Alternatively, purified ORNi-PCR products were subjected to next-generation sequencing (NGS) analysis.

gDNA was extracted from Ba/F3, 293 T, HCT116, and NCI-H1975 cells by standard phenol/chloroform extraction. ORNi-PCR was performed with KOD-Plus-Ver. 2 (Toyobo) in mixtures containing 20 pg–20 ng of gDNA, 0.3 µM of each primer and 0.5–2 µM ORN prepared in a 10 μl volume according to the manufacturer’s protocol using Mastercycler pro S (Eppendorf). For ORNi-PCR using standard three-step cycles, reactions were carried out with an initial denaturation at 94 °C for 2 min, followed by 30 cycles at 98 °C for 10 s, 53–68 °C for 30 s and 68 °C for 50 s. For Two-Step ORNi-PCR, reactions were carried out with an initial denaturation at 94 °C for 2 min, followed by 30–40 cycles at 98 °C for 10 s and 53–68 °C for 70–80 s. ORNi-PCR products were electrophoresed on 1% or 2% agarose gels and, where necessary, subjected to DNA sequencing. DNA sequencing data were analysed using Applied Biosystems Sequence Scanner Software v2.0 (Thermo Fisher Scientific).

RPA was performed using a TwistAmp™ Basic kit (TwistDx, Maidenhead, UK) as follows: a freeze-dried component was reconstituted with 29.5 µl of rehydration buffer and 2.5 µl of each primer (10 µM). A 13.6 µl aliquot was mixed with DNA (20 ng unless stated) to create a pre-reaction mixture. The required blocking agent (0.25–4 µM ORN, 1 µl of CRISPRi RNP complex, 20 or 40 ng of LexA, 0.25–2 µg of MBD2 from the EpiXplore™ Methylated DNA Enrichment Kit (Takara Bio, Shiga, Japan), or 0.5–2 µM 2’3’ddC-modified ODN) and nuclease-free water were added to the pre-reaction mixture to a final volume of 19 µl and incubated at 37 °C for 5 min (pre-incubation). Thereafter, 1 µl of MgOAc (280 mM) was added and the reaction was incubated at 37 °C for a further 30 min. RPA products were purified using a PCR/Gel DNA purification kit (Nippon Genetics, Tokyo, Japan), electrophoresed on 2% or 3% agarose gels, and sequenced if required. DNA gel images were acquired using AE-6905H Image Saver HR (ATTO, Tokyo, Japan) and the DigiDoc-It system (UVP, Cambridge, UK). DNA sequencing data were analyzed using Applied Biosystems Sequence Scanner Software v2.0 (ThermoFisher Scientific, Waltham, MA, USA).