12. Incubate at 37
C for 15 min, followed by a 50
C incubation
for 5 min.
13. Briefly spin down droplets.
14. Place on magnetic rack for 2 min. Carefully transfer the supernatant into a new tube.
15. Place the new tube with the supernatant on the magnetic rack
for 1 min to get rid of trace amounts of beads (optional, see
Note 5).
16. Carefully transfer the supernatant to a new tube (optional, see
Note 5).
17. Add ~180 μL of Agencourt beads (1.8Â RNA volume) to the
total volume of depleted RNA and mix thoroughly by pipetting up and down ~10 times or vortex for 30 s.
18. Incubate the plate at room temp for 10 min.
19. Place the plate on the magnet and wait for 5–10 min allowing
separation of beads from solution.
20. Remove and discard supernatant without disturbing the beads.
21. Add 200 μL of 70% ethanol to each well and incubate for 30 s
at room temp.
22. Remove the ethanol and discard. Repeat two times for a total of
three washes.
23. Let the reaction plate air-dry and uncover for 10 min. Over
drying the sample may result in lower recovery.
24. Elute with 40 μL of 0.1Â TE pH 8.
25. If you need to stop at this point in the protocol, samples can be
stored at À80
C.
3.2 RNAseq Library
Preparation
Recommended input amount: 20 ng to 1 μg mRNA, quantified by
Nanodrop (see Note 6).
1. Assemble the fragmentation and priming reaction on ice in a
nuclease-free tube or PCR Plate and mix by gentle pipetting
(total volume of 10 μL) (see Note 7):
(a) Ribosomal RNA depleted RNA (5 μL).
(b) NEBNext First Strand Synthesis Reaction Buffer (5Â)
(4 μL).
(c) Random Primers (1 μL).
2. Incubate the sample at 94
C (15 min for intact RNA, RNA
Integrity Number > 7 for fragment sizes of approximately
200 bases).
3. Transfer the tube to ICE and proceed immediately to step 4.
RNAseq Workflow
491
C for 15 min, followed by a 50
C incubation
for 5 min.
13. Briefly spin down droplets.
14. Place on magnetic rack for 2 min. Carefully transfer the supernatant into a new tube.
15. Place the new tube with the supernatant on the magnetic rack
for 1 min to get rid of trace amounts of beads (optional, see
Note 5).
16. Carefully transfer the supernatant to a new tube (optional, see
Note 5).
17. Add ~180 μL of Agencourt beads (1.8Â RNA volume) to the
total volume of depleted RNA and mix thoroughly by pipetting up and down ~10 times or vortex for 30 s.
18. Incubate the plate at room temp for 10 min.
19. Place the plate on the magnet and wait for 5–10 min allowing
separation of beads from solution.
20. Remove and discard supernatant without disturbing the beads.
21. Add 200 μL of 70% ethanol to each well and incubate for 30 s
at room temp.
22. Remove the ethanol and discard. Repeat two times for a total of
three washes.
23. Let the reaction plate air-dry and uncover for 10 min. Over
drying the sample may result in lower recovery.
24. Elute with 40 μL of 0.1Â TE pH 8.
25. If you need to stop at this point in the protocol, samples can be
stored at À80
C.
3.2 RNAseq Library
Preparation
Recommended input amount: 20 ng to 1 μg mRNA, quantified by
Nanodrop (see Note 6).
1. Assemble the fragmentation and priming reaction on ice in a
nuclease-free tube or PCR Plate and mix by gentle pipetting
(total volume of 10 μL) (see Note 7):
(a) Ribosomal RNA depleted RNA (5 μL).
(b) NEBNext First Strand Synthesis Reaction Buffer (5Â)
(4 μL).
(c) Random Primers (1 μL).
2. Incubate the sample at 94
C (15 min for intact RNA, RNA
Integrity Number > 7 for fragment sizes of approximately
200 bases).
3. Transfer the tube to ICE and proceed immediately to step 4.
RNAseq Workflow
491