AMPLICON of SARS-CoV-2: adult female
1. Enzymatic fragmentation: PCR product dilution in ultrapure water to 25 ng in 8.75 ul solution, followed by addition of 1.25 ul fragmentation buffer and 2.5 ul fragmentation enzyme, incubating for 5 min at 4C, 20 min at 37C. 2. Library construction: addition of 1.75 ul ER and AT buffer and 0.75 ul enzyme mix to the fragmented solution (volumes per sample), followed by vortexing and incubating for 30 min at 65C with heated lid. The ligation reaction premix was constructed afterwards, using 1.25 ul ultrapure water, 7.5 ul ligation buffer, 2.5 ul DNA ligase, and 1.25 ul adapter stock for marking. Ligation was carried out by incubation for 30 min at 20C. The next step was cleanup with magnetic beads. 3. Library amplification: 10 ul adapter-ligated library and 15 ul PCR mix, 8 PCR cycles according to ABL specifications, followed by cleanup with magnetic beads.