4. To the fragmented and primed mRNA from the previous step,
add the following components and mix by gentle pipetting
(total volume 20 μL):
(a) Fragmented and primed RNA (10 μL).
(b) Nuclease-free water (8 μL).
(c) NEBNext First strand synthesis enzyme mix (2 μL).
5. Incubate the sample in a preheated thermal cycler (with the
heated lid set at !80
C) as follows:
(a) 10 min @ 25
C.
(b) 15 min @ 42
C.
(c) 15 min @ 70
C.
(d) Hold @ 4
C.
6. Proceed directly to second strand cDNA synthesis.
7. Add the following reagents to the First Strand Synthesis reaction (20 μL) and mix thoroughly by gentle pipetting on ice
(total volume of 80 μL):
(a) First Strand Synthesis Product (20 μL).
(b) NEBNext Second Strand Synthesis Reaction Buffer (10Â)
(8 μL).
(c) NEBNext Second Strand Synthesis Enzyme mix (4 μL).
(d) Nuclease-free water (48 μL).
8. Incubate in a thermal cycler for 1 h at 16
C, with heated lid set
at 40
C.
9. Vortex AMPure XP Beads to resuspend and bring to room
temperature (see Note 8).
10. Add 144 μL (1.8Â) of resuspended AMPure XP Beads to the
second strand synthesis reaction (~80 μL). Mix well by pipetting up and down at least ten times.
11. Incubate for 5 min at room temperature.
12. Quickly spin the tube to collect any sample on the sides of the
tube. Place the tube on a magnetic rack. After the solution is
clear (about 5 min), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA
targets.
13. 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.
14. Repeat step 13 once for a total of two washing steps.
15. Air dry the beads for 5 min while tubes are on the magnetic
rack with lid open. Make sure to 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).
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