1.1 Amplify the purified DNA was amplified by primers with sequencing indexes.
1.2 Mix1 μl 10 μM Index-F primer, 1 μl 10 μM Index-R primer, 8 μl ligated DNA, and 10 μl 2X HIFI PCR Master Mix (NEB, Cat. #M0541) and incubate at 72 °C for 5 min, 98 °C for 30 s, 20 cycles of 98 °C for 10 s and 63 °C for 10 s, then 72 °C for 1 min, and hold at 16 °C.
1.3 Purified PCR products by adding 0.9X volume of VAHTS DNA clean beads (VAHTS, Cat. #N411-03), and gently mixing by pipetting, place the tube at RT for 10 min.
1.4 Place the tube on the magnet stand for 1 min, and then remove the liquid.
1.5 Add 200 μl 80% ethanol (fresh prepared) to the tube, keep the tube on the magnet stand, and do not mix or resuspend beads. Remove the ethanol after 30 s.
1.6 Repeat adding 200 μl 80% ethanol (fresh prepared) to the tube, keep the tube on the magnet stand, and do not mix or resuspend beads. Remove the ethanol after 30 s.
1.7 Air dry the beads for about 2-5 min. Do not over-dry the beads.
1.8 Remove samples for the magnet stand.
1.9 Resuspended beads with 20 µl H2O, and incubate at RT for 5 min.
1.10 Place the tube on the magnet stand until the solution turns clear (around 30 s) and take all the liquid containing constructed libraries to the new tube.
1.11 Detect concentrations of the constructed libraries by adding 1 μl DNA to 199 μl Equalbit 1X dsDNA HS Working Solution (Vazyme, Cat. #121-01-AA). Or any preferred method.
1.12 Library sequencing is performed with an Illumina NovaSeq platform with pair-end reads of 150 bp. Or any preferred method.
Index-F primer: AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX TCGTCGGCAGCGTCAGATGTGTAT (XXXXXXXX is the index sequence for sequencing)
Index-R primer: CAAGCAGAAGACGGCATACGAGAT XXXXXXXX GTCTCGTGGGCTCGGAGATGTG (XXXXXXXX is the index sequence for sequencing)