TITLE: Direct cDNA Sequencing (SQK-DCS109)
AUTHORS: e.gustavsson

[DESCRIPTION]
This protocol describes how to prepare direct cDNA Sequencing libraries for nanopore sequencing without using PCR.

[STEPS]
SECTION: Reverse transcription and strand-switching
1. 
 

SECTION: Reverse transcription and strand-switching
2. Prepare the RNA in Nuclease-free water.
SECTION: Reverse transcription and strand-switching
2.1. Transfer 100 ng into a 1.5 ml Eppendorf DNA LoBind tube.

SECTION: Reverse transcription and strand-switching
2.2. Adjust the volume to up to 7.5 µL with Nuclease-free water.

SECTION: Reverse transcription and strand-switching
2.3. Mix by flicking the tube to avoid unwanted shearing.
SECTION: Reverse transcription and strand-switching
2.4. Spin down briefly in a microfuge.
SECTION: Reverse transcription and strand-switching
3. Prepare the following reaction in a 0.2 ml PCR tube: 
x μl poly A+ RNA, 100 ng 
2.5 µL
1 µL 
7.5-x μl RNase-free water


SECTION: Reverse transcription and strand-switching
4. Mix gently by flicking the tube, and spin down.
SECTION: Reverse transcription and strand-switching
5. Incubate at 65 °C for 5 min and then snap cool on a pre-chilled freezer block.

SECTION: Reverse transcription and strand-switching
6. In a separate tube, mix together the following: 
4 µL
1 µL
1 µL
2 µL



SECTION: Reverse transcription and strand-switching
7. Mix gently by flicking the tube, and spin down. 
SECTION: Reverse transcription and strand-switching
8. Add the strand-switching buffer to the snap-cooled, annealed mRNA, mix by flicking the tube and spin down.
SECTION: Reverse transcription and strand-switching
9. Incubate at 42 °C for 2 min.

SECTION: Reverse transcription and strand-switching
10. Add 1 µL. The total volume is now 20 µL.

SECTION: Reverse transcription and strand-switching
11. Mix gently by flicking the tube, and spin down.
SECTION: Reverse transcription and strand-switching
12. Incubate using the following protocol: 
Reverse transcription and strand-switching 90 min @ 42 °C (1 cycle) 
Heat inactivation 5 min @ 85 °C (1 cycle) 
Hold @ 4 °C




SECTION: RNA degradation and second strand synthesis
13. Add 1 µL to the reverse transcription reaction. 

SECTION: RNA degradation and second strand synthesis
14. Incubate the reaction for 10 min at 37 °C.

SECTION: RNA degradation and second strand synthesis
15. Resuspend the AMPure XP beads by vortexing.
SECTION: RNA degradation and second strand synthesis
16. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: RNA degradation and second strand synthesis
17. Add 17 µL to the reaction and mix by flicking the tube.

SECTION: RNA degradation and second strand synthesis
18. Incubate on a Hula mixer (rotator mixer) for 5 min at 37 Room temperature.

SECTION: RNA degradation and second strand synthesis
19. Prepare 500 µL fresh 70 %ethanol in Nuclease-free water.

SECTION: RNA degradation and second strand synthesis
20. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: RNA degradation and second strand synthesis
21. Keep the tube on the magnet and wash the beads with 200 µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: RNA degradation and second strand synthesis
22. Repeat the previous step.
SECTION: RNA degradation and second strand synthesis
23. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 s, but do not dry the pellet to the point of cracking.

SECTION: RNA degradation and second strand synthesis
24. Remove the tube from the magnetic rack and resuspend pellet in 20 µL.

SECTION: RNA degradation and second strand synthesis
25. Incubate on a Hula mixer (rotator mixer) for 10 min at 37 Room temperature.

SECTION: RNA degradation and second strand synthesis
26. Pellet beads on magnet until the eluate is clear and colourless.
SECTION: RNA degradation and second strand synthesis
27. Remove and retain 20 µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: RNA degradation and second strand synthesis
28. Prepare the following reaction in a 0.2 ml thin-walled PCR tube: 
25 µL
2 µL
20 µL
3 µL



SECTION: RNA degradation and second strand synthesis
29. Incubate using the following protocol: 
94 °C 1 min 1 
50 °C 1 min 1 
65 °C 15 min 1 
4 °C ∞






SECTION: RNA degradation and second strand synthesis
30. Resuspend the AMPure XP beads by vortexing.
SECTION: RNA degradation and second strand synthesis
31. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: RNA degradation and second strand synthesis
32. Add 40 µL to the reaction and mix by flicking the tube.

SECTION: RNA degradation and second strand synthesis
33. Incubate on a Hula mixer (rotator mixer) for 5 min at 4 Room temperature.

SECTION: RNA degradation and second strand synthesis
34. Prepare 500 µL.

SECTION: RNA degradation and second strand synthesis
35. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: RNA degradation and second strand synthesis
36. Keep the tube on the magnet and wash the beads with 200 µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: RNA degradation and second strand synthesis
37. Repeat the previous step.
SECTION: RNA degradation and second strand synthesis
38. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 s, but do not dry the pellet to the point of cracking.

SECTION: RNA degradation and second strand synthesis
39. Remove the tube from the magnetic rack and resuspend pellet in 21 µL.

SECTION: RNA degradation and second strand synthesis
40. Incubate on a Hula mixer (rotator mixer) for 10 min at 4 Room temperature.

SECTION: RNA degradation and second strand synthesis
41. Pellet beads on magnet until the eluate is clear and colourless.
SECTION: RNA degradation and second strand synthesis
42. Remove and retain 21 µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: RNA degradation and second strand synthesis
43. Analyse 1 µL for size, quantity and quality.

SECTION: End-prep
44. 
 

SECTION: End-prep
45. Perform end repair and dA-tailing of fragmented DNA as follows: 
20 µL
30 µL
7 µL 
3 µL



SECTION: End-prep
46. Mix gently by pipetting and spin down.
SECTION: End-prep
47. Using a thermal cycler, incubate at 20 °C for 5 min and 65 °C for 5 min.

SECTION: End-prep
48. Resuspend the AMPure XP beads by vortexing.
SECTION: End-prep
49. Transfer the sample to a 1.5 ml DNA LoBind Eppendorf tube.
SECTION: End-prep
50. Add 60 µL to the end-prep reaction and mix by pipetting.

SECTION: End-prep
51. Incubate on a Hula mixer (rotator mixer) for 5 min at 65 Room temperature.

SECTION: End-prep
52. Prepare 500 µL.

SECTION: End-prep
53. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: End-prep
54. Keep the tube on the magnet and wash the beads with 200 µL without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: End-prep
55. Repeat the previous step.
SECTION: End-prep
56. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 s, but do not dry the pellet to the point of cracking.

SECTION: End-prep
57. Remove the tube from the magnetic rack and resuspend pellet in 30 µL. Incubate for 2 min at 65 Room temperature.

SECTION: End-prep
58. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: End-prep
59. Remove and retain 30 µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: End-prep
60. Take forward 30 µL into adapter ligation.

SECTION: Adapter ligation
61. Check the contents of each tube are clear of any precipitate and are thoroughly mixed before setting up the reaction.
SECTION: Adapter ligation
61.1. Mix the contents of each tube by flicking.
SECTION: Adapter ligation
61.2. Check that there is no precipitate present (DTT in the Blunt/TA Master Mix can sometimes form a precipitate).
SECTION: Adapter ligation
61.3. Spin down briefly before accurately pipetting the contents in the reaction.
SECTION: Adapter ligation
62. Taking the end-prepped DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition. 
30 µL
5 µL  
50 µL
15 µL



SECTION: Adapter ligation
63. Mix gently by flicking the tube, and spin down.
SECTION: Adapter ligation
64. Incubate the reaction for 10 min at 65 Room temperature.

SECTION: AMPure XP bead binding
65. Resuspend the AMPure XP beads by vortexing.
SECTION: AMPure XP bead binding
66. Add 40 µL to the adapter ligation reaction from the previous step and mix by pipetting.

SECTION: AMPure XP bead binding
67. Incubate on a Hula mixer (rotator mixer) for 5 min at 65 Room temperature.

SECTION: AMPure XP bead binding
68. Place on magnetic rack, allow beads to pellet and pipette off supernatant.
SECTION: AMPure XP bead binding
69. Add 200 µL to the beads. Resuspend the beads by pipetting up and down. Return the tube to the magnetic rack, allow beads to pellet and pipette off the supernatant.

SECTION: AMPure XP bead binding
70. Repeat the previous step.
SECTION: AMPure XP bead binding
71. Spin down and place the tube back on the magnet. Pipette off any residual supernatant.
SECTION: AMPure XP bead binding
72. Remove the tube from the magnetic rack and resuspend pellet in 25 µL.

SECTION: AMPure XP bead binding
73. Incubate on a Hula mixer (rotator mixer) for 10 min at 65 Room temperature.

SECTION: AMPure XP bead binding
74. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: AMPure XP bead binding
75. Remove and retain 25 µL into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: AMPure XP bead binding
76. Quantify 1 µL using a Qubit fluorometer - recovery aim ~60 fmol.

SECTION: Priming and loading the flow cell
77. 
 

 

SECTION: Priming and loading the flow cell
78. Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at 65 Room temperature.

SECTION: Priming and loading the flow cell
79. Mix the Sequencing Buffer (SQB), Flush Buffer (FB) and Flush Tether (FLT) tubes by vortexing and spin down at 65 Room temperature.

SECTION: Priming and loading the flow cell
80. To prepare the flow cell priming mix, add 30 µL directly to 1 tube of thawed and mixed Flush Buffer (FB), and mix by vortexing.

SECTION: Priming and loading the flow cell
81. Load the flow cell(s) into the docking ports within the PromethION.
SECTION: Priming and loading the flow cell
82. Prime the flow cell using the following steps, taking care to avoid the introduction of air bubbles.
SECTION: Priming and loading the flow cell
82.1. Turn the valve to expose the inlet port (Port 1).
SECTION: Priming and loading the flow cell
82.2. A small tract of air will be visible beyond the inlet port. Draw back a small volume to remove any air bubbles (a few µls): 

a. Set a P1000 pipette to 200 µl 
b. Insert the tip into the inlet port 
c. Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.
SECTION: Priming and loading the flow cell
82.3. Using a P1000 pipette, flush 500 µL into the inlet port of the flow cell, avoiding the introduction of air bubbles.

SECTION: Priming and loading the flow cell
82.4. Wait 5 min. During this time you can prepare your library for loading, as described in the next steps.

SECTION: Priming and loading the flow cell
82.5. Repeat the priming step with another 500 µL.

SECTION: Priming and loading the flow cell
83. Thoroughly mix the contents of the Loading Beads (LB) tubes by vortexing.
SECTION: Priming and loading the flow cell
84. In a new tube, prepare the library for loading as follows: 
75 µL
51 µL
24 µL 


SECTION: Priming and loading the flow cell
85. Load your sample.
SECTION: Priming and loading the flow cell
85.1. Load 150 µL through the inlet port.

SECTION: Priming and loading the flow cell
85.2. Close the valve to seal the inlet port and close the PromethION lid when ready.
SECTION: Priming and loading the flow cell
85.3. Wait a minimum of 10 min after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

SECTION: Ending the experiment
86. After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Wash Kit instructions and store the washed flow cell at 2 °C - 8 °C, OR

SECTION: Ending the experiment
87. Follow the returns procedure by washing out the flow cell ready to send back to Oxford Nanopore.
SECTION: Ending the experiment
88.