16. Remove the tube from the magnet. Elute the DNA target from
the beads into 53 μL of 0.1Â TE Buffer provided in the kit.
Mix well on a vortex mixer or by pipetting up and down.
Quickly spin the tube and incubate for 2 min at room temperature. Place the tube in the magnetic rack until the solution is
clear.
17. Remove 50 μL of the supernatant and transfer to a clean
nuclease-free PCR tube or plate.
18. If you need to stop at this point in the protocol, samples can be
stored at À20
C before proceeding to library construction
(steps 19–34).
19. Add the following reagents to the supernatant from step 17 on
ICE and vortex gently to mix (total volume of 60 μL):
(a) Second Strand cDNA synthesis product (50 μL).
(b) NEBNext Ultra II End Prep reaction Buffer (7 μL).
(c) NEBNext Ultra II End prep enzyme mix (3 μL).
20. Incubate the sample in a thermocycler (with the heated lid set
at 75
C) as follows:
(a) 30 min @ 20
C.
(b) 30 min @ 65
C.
(c) Hold @ 4
C.
21. Proceed immediately to Adaptor Ligation (steps 22–34).
22. Dilute the NEBNext Adaptor prior to setting up the ligation
reaction in ice-cold Adaptor Dilution Buffer and keep the
diluted adaptors on ice. If the purified RNA is between 1 and
10 ng, dilute 25-fold in Adaptor Dilution Buffer, if between
11 and 100 ng, then dilute 5-fold in Adaptor Dilution Buffer.
23. Add the following components directly to the End Prep Reaction (volumes given are for one library, resulting in a total
volume of 93.5 μL). Do not premix the components to
prevent adaptor-dimer formation (see Note 10). The ligation enhancer and ligation master mix may be premixed.
(a) End prepped DNA (60 μL).
(b) Diluted Adaptor (2.5 μL).
(c) NEBNext Ligation Enhancer (1 μL).
(d) NEBNext Ultra II Ligation Master Mix (30 μL).
24. Mix by pipetting followed by a quick spin to collect all liquid
from the sides of the tube.
25. Incubate for 15 min at 20
C in a thermal cycler.
26. Add 3 μL of USER Enzyme to the ligation mixture from step
25. Mix well and incubate at 37
C for 15 min.
RNAseq Workflow
493
the beads into 53 μL of 0.1Â TE Buffer provided in the kit.
Mix well on a vortex mixer or by pipetting up and down.
Quickly spin the tube and incubate for 2 min at room temperature. Place the tube in the magnetic rack until the solution is
clear.
17. Remove 50 μL of the supernatant and transfer to a clean
nuclease-free PCR tube or plate.
18. If you need to stop at this point in the protocol, samples can be
stored at À20
C before proceeding to library construction
(steps 19–34).
19. Add the following reagents to the supernatant from step 17 on
ICE and vortex gently to mix (total volume of 60 μL):
(a) Second Strand cDNA synthesis product (50 μL).
(b) NEBNext Ultra II End Prep reaction Buffer (7 μL).
(c) NEBNext Ultra II End prep enzyme mix (3 μL).
20. Incubate the sample in a thermocycler (with the heated lid set
at 75
C) as follows:
(a) 30 min @ 20
C.
(b) 30 min @ 65
C.
(c) Hold @ 4
C.
21. Proceed immediately to Adaptor Ligation (steps 22–34).
22. Dilute the NEBNext Adaptor prior to setting up the ligation
reaction in ice-cold Adaptor Dilution Buffer and keep the
diluted adaptors on ice. If the purified RNA is between 1 and
10 ng, dilute 25-fold in Adaptor Dilution Buffer, if between
11 and 100 ng, then dilute 5-fold in Adaptor Dilution Buffer.
23. Add the following components directly to the End Prep Reaction (volumes given are for one library, resulting in a total
volume of 93.5 μL). Do not premix the components to
prevent adaptor-dimer formation (see Note 10). The ligation enhancer and ligation master mix may be premixed.
(a) End prepped DNA (60 μL).
(b) Diluted Adaptor (2.5 μL).
(c) NEBNext Ligation Enhancer (1 μL).
(d) NEBNext Ultra II Ligation Master Mix (30 μL).
24. Mix by pipetting followed by a quick spin to collect all liquid
from the sides of the tube.
25. Incubate for 15 min at 20
C in a thermal cycler.
26. Add 3 μL of USER Enzyme to the ligation mixture from step
25. Mix well and incubate at 37
C for 15 min.
RNAseq Workflow
493