Fragment DNA to the appropriate size range and adjust the sample volume to 50 µl containing 500 pg to 1 µg of fragmented DNA. Ensure all kit reagents are well mixed before use.
▶ 00:02
2
Perform end repair and dA-tailing
Add 3 µl of End Prep Enzyme Mix and 7 µl of End Prep Reaction Buffer to the 50 µl fragmented DNA sample. Incubate at 20°C for 20 minutes, then 65°C for 20 minutes, and hold at 4°C.
▶ 01:16
3
Ligate adaptors to DNA fragments
Add Ultra II Ligation Master Mix, Ligation Enhancer, and Adaptor to the End Prep reaction and mix thoroughly. Incubate at 20°C for 15 minutes with heated lid off, then add USER enzyme and incubate at 37°C for 15 minutes.
▶ 02:18
4
Perform size selection with magnetic beads
Conduct two rounds of magnetic bead-based size selection: the first round removes large DNA fragments and the second removes small DNA fragments. Wash the beads with 80% ethanol, allow to air dry, and elute the library in 17 µl of Tris-HCl or 0.1X TE Buffer.
▶ 03:57
5
Amplify library by PCR
Add universal PCR primer, index primer, and Ultra II Q5 Master Mix to the 15 µl eluted library sample. Run PCR with the appropriate number of cycles based on the original input DNA amount.
▶ 07:17
6
Purify PCR product using magnetic beads
Add 45 µl of vortexed magnetic beads to the PCR reaction and incubate for 5 minutes. Remove supernatant, wash beads with 80% ethanol, allow to air dry, and elute the final library in 33 µl of 0.1X TE Buffer.
▶ 08:05
7
Verify and quantitate final library
Confirm the size distribution of the library by running a diluted sample on a Bioanalyzer using the High-Sensitivity Chip. Quantitate the library using qPCR-based methods or electrophoretic methods.