In this study, we first probed the enzymatic digestions that have been most utilized in literature for post-ligation purification (Figure ). These were utilized for NSA detection for self-annealing ligation (cDNA 1C), splint-padlock ligation (cDNA 4C), and cohesive end ligation (cDNA 11C). These included single enzyme (either Exo I or III) treatment with respective buffers (conditions C1 and C2) and no exonuclease digestion (conditions C4, Table ). Besides, dual exonuclease digestion in T4 DNA ligase buffer was also investigated (condition C3, Table ). After treatment, the substrates were subjected to an RCA amplification mix either in the absence (for quantifying NSA) or presence of primer (as positive control and for normalization) with fluorescence measurement. The end-RFU and NNSA quantifications have been enumerated in Table .