27. Add 87 μL (0.9Â) of resuspended AMPure XP Beads and mix
well on a vortex mixer or by pipetting up and down at least ten
times.
28. Incubate for 5 min at room temperature.
29. Quickly spin the tube in a microcentrifuge and place the tube
on an appropriate magnetic rack to separate beads from the
supernatant. After the solution is clear (about 5 min), discard
the supernatant that contains unwanted fragments (do not
discard the beads).
30. Add 200 μL of freshly prepared 80% ethanol to the tube while
in the magnetic rack. Incubate at room temperature for 30 s,
then carefully remove, and discard the supernatant.
31. Repeat step 30 once for a total of two washing steps.
32. Air-dry beads for 5 min while the tube is on the magnetic rack
with the lid open. Remove any residual ethanol after 5 min. Do
not over dry the beads. This may result in lower recovery of
DNA target (see Note 9).
33. Remove the tube from the magnet. Elute DNA target from the
beads with 17 μL of 0.1Â TE provided in the kit. Mix well on a
vortex mixer or by pipetting up and down and incubate for
2 min at room temperature. Put the tube in the magnetic rack
until the solution is clear.
34. Without disturbing the bead pellet, transfer 15 μL of the
supernatant to a clean PCR tube and proceed to PCR
enrichment.
35. If you need to stop at this point in the protocol, samples
can be stored at À20 ˚C before proceeding to multiplexing
libraries and PCR enrichment of adaptor-ligated DNA
(steps 36–47).
36. To the cDNA (15 μL) from step 34, add the following components and mix by gentle pipetting (see Notes 11 and 12) for
a total volume of 50 μL:
(a) Adaptor Ligated DNA (15 μL).
(b) NEBNext Ultra II Q5 Master Mix (25 μL).
(c) Universal PCR Primer/i5 Primer (5 μL).
(d) Index (X) Primer/i7 Primer (5 μL).
37. Gently pipette up and down 5–10 times to mix.
38. Set up the following PCR Cycling Conditions (see Notes 13
and 14):
(a) Initial denaturation: 98
C, 30 s, 1 cycle.
(b) Denaturation Annealing/Extension: 98
C, 10 s followed
by 65
C for 75 s, 5–14 cycles.
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Shuyi Ma et al.
well on a vortex mixer or by pipetting up and down at least ten
times.
28. Incubate for 5 min at room temperature.
29. Quickly spin the tube in a microcentrifuge and place the tube
on an appropriate magnetic rack to separate beads from the
supernatant. After the solution is clear (about 5 min), discard
the supernatant that contains unwanted fragments (do not
discard the beads).
30. Add 200 μL of freshly prepared 80% ethanol to the tube while
in the magnetic rack. Incubate at room temperature for 30 s,
then carefully remove, and discard the supernatant.
31. Repeat step 30 once for a total of two washing steps.
32. Air-dry beads for 5 min while the tube is on the magnetic rack
with the lid open. Remove any residual ethanol after 5 min. Do
not over dry the beads. This may result in lower recovery of
DNA target (see Note 9).
33. Remove the tube from the magnet. Elute DNA target from the
beads with 17 μL of 0.1Â TE provided in the kit. Mix well on a
vortex mixer or by pipetting up and down and incubate for
2 min at room temperature. Put the tube in the magnetic rack
until the solution is clear.
34. Without disturbing the bead pellet, transfer 15 μL of the
supernatant to a clean PCR tube and proceed to PCR
enrichment.
35. If you need to stop at this point in the protocol, samples
can be stored at À20 ˚C before proceeding to multiplexing
libraries and PCR enrichment of adaptor-ligated DNA
(steps 36–47).
36. To the cDNA (15 μL) from step 34, add the following components and mix by gentle pipetting (see Notes 11 and 12) for
a total volume of 50 μL:
(a) Adaptor Ligated DNA (15 μL).
(b) NEBNext Ultra II Q5 Master Mix (25 μL).
(c) Universal PCR Primer/i5 Primer (5 μL).
(d) Index (X) Primer/i7 Primer (5 μL).
37. Gently pipette up and down 5–10 times to mix.
38. Set up the following PCR Cycling Conditions (see Notes 13
and 14):
(a) Initial denaturation: 98
C, 30 s, 1 cycle.
(b) Denaturation Annealing/Extension: 98
C, 10 s followed
by 65
C for 75 s, 5–14 cycles.
494
Shuyi Ma et al.