Nebnext da tailing kit

DNA (2 μg) was resuspended in 50 μL distilled water and sheared to approximately 550-bp fragments using an S220 focused ultrasonicator (Covaris) according to the manufacturer’s protocol. Fragmented DNA was repaired using NEBNext blunt-end repair kit (New England Biolabs) and purified using Monarch PCR cleanup kit (NEB). Blunted DNA was A-tailed using NEBNext dA-tailing kit (NEB) and column purified. Custom transposon sequencing adaptors (Table S4) were generated by heating an equimolar mix of Com_AdaptorPt1 primer and Com_AdaptorPt2 (P7+index) primers to 95°C for 5 min, followed by cooling by 1°C every 40 s to a final temperature of 4°C in a thermocycler. Adaptors were ligated to A-tailed library fragments using NEBNext quick ligase kit. Transposon-containing fragments were enriched by PCR using the ComP7 primer (10 μM) and an equimolar mix of primers P5-IR2a-d primer (10 μM) in a reaction with 50 ng of adaptor-ligated template and Phusion DNA polymerase (NEB) in a thermocycler with the following program: 98°C for 3 min; 4 cycles of 98°C for 20 s, 70°C 20 for s, and 72°C for 1 min; 20 cycles of 98°C for 20 s, 67°C for 20 s, and 72°C for 1 min; and 72°C for 3 min. Transposon-enriched libraries were subsequently purified with AMPure XP beads (Beckman), pooled, and further purified using AMPure XP beads.