RAP-DNA Sequencing Library Preparation
Goal:
Prepare Illumina sequencing libraries from co-purified DNA. This protocol uses the
NEBNext Ultra Library Prep Kit for Illumina and the NEBNext Multiplex Oligos for
Illumina, with modifications we designed for making libraries from small amounts of
short-fragment DNA.
1. Start with 12.5 µL of purified DNA in H2O in low-retention PCR strip tubes.
2. Add 2.5 µL of master mix containing 1.5 µL 10× NEBNext End Repair Reaction Buffer and
1 µL NEBNext End Prep Enzyme Mix.
3. Mix by pipet and incubate at 20°C for 30 minutes.
4. Add 1 µL of 160 mM NaCl (15 mM final concentration) and mix.
5. Incubate at 55°C for 30 minutes, then hold at 4°C.
6. To the end repair reaction, add 1 µL of a 1:10 dilution of NEBNext Adaptor for Illumina.
Then add 4 µL of master mix containing 3.75 µL of Blunt/TA Ligase Master Mix and 0.25
µL NEBNext Ligation Enhancer.
7. Mix and incubate at 20°C for 45 minutes.
8. Add 1 µL of USER enzyme. Mix and incubate at 37°C for 15 minutes.
9. Add 19 µL H2O to 40 µL total volume.
10. Clean once using 0.7× volume (28 µL) SPRI beads. At end, add 40 µL H2O but do not
remove from beads.
11. Clean again use 1× volume (40 µL) SPRI beads. At end, elute in 24 µL and remove 23 µL
from the beads.
12. Set up PCR reaction:
DNA
NEBNext Indexed PCR Primer (25 µM)
NEBNext Unversal PCR Primer (25 µM)
NEBNext High-Fidelity 2× Master Mix (NEB)
Total
23
1
1
25
50
µl
µl
µl
µl
µl
13. Run the following PCR program:
Initial Denaturation
Denaturation
Annealing
Extension
Denaturation
Annealing and Extension
Final Extension
Hold
98°C
98°C
67°C
72°C
98°C
72°C
72°C
4°C
30 seconds
10 seconds
30 seconds
30 seconds
10 seconds
30 seconds
60 seconds
hold
1 cycle
4 cycles
4-10* cycles
1 cycle
*RAP samples usually require 10 cycles during this step, while inputs require between 4 and
6.
14. Clean once using 1× volume (50 µL) SPRI beads. At end, add 50 µL H2O but do not remove
from beads.
15. Clean again use 1× volume (50 µL) SPRI beads. At end, elute in 13 µL H2O.
16. Measure library concentration with Qubit fluorometric quantitation.
17. Examine DNA fragment sizes using the High-Sensitivity DNA Bioanalyzer kit.
18. Pool multiple barcoded libraries and sequence with Illumina.