Circular DNA obtained by the circularization reaction was combined with 12 μl 5× Annealing buffer (50 mM Tris @ pH 7.5–8.0, 250 mM NaCl, 5 mM EDTA) and 1 μl Exo-resistant random primers (Thermofisher), heated for 5 min at 98 °C and then cooled down at room temperature. Subsequently, the RCA mix (previous reaction mixture, 10 μl 10× Phi29 Buffer (Thermofisher), 2 μl BSA (New England Biolabs), 10 μl dNTPs (Thermofisher), 4 μl pyrophosphatase (Thermofisher), 2 μl Phi29 Polymerase (Thermofisher), and MQ (to a volume of 100 μl)) was prepared. RCA was performed overnight at 30 °C. The RCA-reaction was inactivated by 10 min incubation at 70 °C.
To test whether CyclomicsSeq worked, 4 μl of RCA mixture was incubated with a restriction enzyme that specifically cuts backbone-backbone interactions, but not backbone-insert interactions. Briefly, 4 μl of RCA mixture was combined with 4 μl Restriction enzyme buffer (New England Biolabs), 13 μl MilliQ, and 1 μl BglII (New England Biolabs). The reaction mixture was incubated for 1 h at 37 °C and then ran on a 1.5% Agarose gel.
To test whether CyclomicsSeq worked, 4 μl of RCA mixture was incubated with a restriction enzyme that specifically cuts backbone-backbone interactions, but not backbone-insert interactions. Briefly, 4 μl of RCA mixture was combined with 4 μl Restriction enzyme buffer (New England Biolabs), 13 μl MilliQ, and 1 μl BglII (New England Biolabs). The reaction mixture was incubated for 1 h at 37 °C and then ran on a 1.5% Agarose gel.