RAP-DNA Sequencing Library Preparation. [ download protocol ]

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

    23ul

    NEBNext Indexed PCR Primer (25 µM)

    1ul

    NEBNext Unversal PCR Primer (25 µM)

    1ul

    NEBNext High-Fidelity 2× Master Mix (NEB)

    25ul

    Total

    50ul

  1. Run the following PCR program:
    Initial Denaturation 98°C

    30 seconds

    1 cycle

    Denaturation

    98°C

    10 seconds

     

    Annealing

    67°C

    30 seconds

    4 cycles

    Extension

    72°C

    30 seconds

     
    Denaturation 98°C 10 seconds 4-10* cycles
    Annealing and Extension 72°C 30 seconds  
    Final Extension 72°C 60 seconds 1 cycle
    Hold 4°C hold  
  1. *RAP samples usually require 10 cycles during this step, while inputs require between 4 and 6.
  2. Clean once using 1× volume (50 µL) SPRI beads. At end, add 50 µL H2O but do not remove from beads.
  3. Clean again use 1× volume (50 µL) SPRI beads. At end, elute in 13 µL H2O.
  4. Measure library concentration with Qubit fluorometric quantitation.
  5. Examine DNA fragment sizes using the High-Sensitivity DNA Bioanalyzer kit.
  6. Pool multiple barcoded libraries and sequence with Illumina.