TITLE: Nanopore SISPA 
AUTHORS: Siu Fung Ho

[DESCRIPTION]
Nanopore SISPA

[STEPS]
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
3. Add 1:500 linear acrylamide instead of carrier RNA to Buffer AVL.
SECTION: Sample Preprocessing
2. Use 280 μl of the filtrate as starting material for the Qiagen Viral RNA Mini Kit.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
4. Pipet 1120 μL prepared Buffer AVL into a 2 mL Eppendorf tube.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
5. Add 280 μL of sample into the tube containing Buffer AVL. 
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
7. Incubate at room temperature for 10 min.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
8. Briefly centrifuge the tube to remove drops from the inside of the lid.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
9. Add 1120 μL 100% ethanol to the sample, and mix by pulse-vortexing for 15 s. 
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
6. Mix by pulse-vortexing for 15 s.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
10. After mixing, briefly centrifuge the tube to remove drops from inside the lid.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
11. Carefully apply 630 μL of the lysate to the QIAamp Mini column without wetting the rim. Close the cap, and centrifuge at 6000 x g for 1 min. 

Place the QIAamp Mini column into a clean 2 mL collection tube, and discard the tube containing the filtrate.

 

SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
12. Repeat the previous step until all of the lysate has been added to the column.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
13. Carefully open the QIAamp Mini column, and add 500 μL Buffer AW1. 

Close the cap, and centrifuge at 6000 x g for 1 min. 

Place the QIAamp Mini column in a clean 2 mL collection tube, and discard the tube containing the filtrate.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
14. Carefully open the QIAamp Mini column, and add 500 μL Buffer AW2. 

Close the cap and centrifuge at 16,000 x g  for 3 min.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
15. Place the QIAamp Mini column in a new 2 mL collection tube, and discard the old collection tube with the filtrate. 

Centrifuge at 16,000 x g for 1 min.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
16. Place the QIAamp Mini column in a clean 1.5 mL microcentrifuge tube. Discard the old collection tube containing the filtrate. 

Carefully open the QIAamp Mini column and add 50 μL Buffer AVE equilibrated to room temperature. 

Close the cap, and incubate at room temperature for 1 min.
SECTION: RNA extraction with QIAamp Viral RNA Mini Kit
17. Centrifuge at 6000 x g (8000 rpm) for 1 min to collect eluate.
SECTION: DNase treatment and RNA cocentration
18. Mix 50 μL eluate with  100 μL of RNA Binding Buffer.
SECTION: DNase treatment and RNA cocentration
19. Add and mix in 150 μL of 100% ethanol.
SECTION: DNase treatment and RNA cocentration
20. Transfer the sample to the Zymo-Spin IC Column3 in a CollectionTube and centrifuge. 

Discard the flow-through
SECTION: DNase treatment and RNA cocentration
21. Add 400 μl RNA Wash Buffer to the column, centrifuge and discard the flow-through.
SECTION: DNase treatment and RNA cocentration
22. Make up a DNase I Reaction Mix for each sample to be treated:

Component                           Volume
DNase I                           5 µL
DNA Digestion Buffer    35 µL

SECTION: DNase treatment and RNA cocentration
23. Add 40 μl of DNase I Reaction Mix directly into column matrix and incubate at room temperature for 15 minutes.
SECTION: DNase treatment and RNA cocentration
24. Add 700 μl RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
SECTION: DNase treatment and RNA cocentration
25. Add 400 μl RNA Wash Buffer to the column and centrifuge for 1 minute ensure complete removal of the wash buffer. 

Carefully, transfer the column into a RNase-free tube.
SECTION: DNase treatment and RNA cocentration
26. Add 10 μl DNase/RNase-Free Water directly to the column matrix and centrifuge.
 

SECTION: Reverse Transcription
45. Prepare RT reaction master mix. Per sample volumes:

Component                             Volume
DNase I treated RNA       6 µL
dNTP 10mM                     0.5 µL
SISPA-A primer 40uM     1 µL
Nuclease-free water       2.5 µL

SECTION: Reverse Transcription
47. Incubate at 65C for 5min and then on ice for 1 min
SECTION: Reverse Transcription
46. Mix and briefly centrifuge.
SECTION: Reverse Transcription
48. Pre-warm the 5x SSIV buffer to RT. Vortex and briefly centrifuge.
SECTION: Second Strand cDNA Synthesis (Klenow fragment)
52. Add 1ul of 10x NEB buffer 2 to the RT product.
SECTION: Second Strand cDNA Synthesis (Klenow fragment)
53. Incubate at 95C for 3 min and then one ice for 1min.
SECTION: Second Strand cDNA Synthesis (Klenow fragment)
54. Add 0.5ul of Klenow fragment to the mixture.
SECTION: Second Strand cDNA Synthesis (Klenow fragment)
55. Incubate at 37C for 1 h and then at 75C for 15 min.
SECTION: AMPure cleanup
56. Prepare the AMPure XP beads for use; resuspend by vortexing.
SECTION: AMPure cleanup
57. Transfer the cDNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: AMPure cleanup
58. Add 11 μl of resuspended AMPure XP beads to the cDNA samples and mix by pipetting up and down.
SECTION: AMPure cleanup
59. Incubate for 5 minutes at room temperature.
SECTION: AMPure cleanup
60. Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
SECTION: AMPure cleanup
61. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: AMPure cleanup
62. Keep on magnet, wash beads with 200 μl of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard.
SECTION: AMPure cleanup
63. Repeat the previous step.
SECTION: AMPure cleanup
64. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 3 min, but do not dry the pellet to the point of cracking.
SECTION: AMPure cleanup
65. Remove the tube from the magnetic rack and resuspend pellet in 10 μl nuclease-free water. Incubate for 2 minutes at room temperature.
SECTION: AMPure cleanup
66. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: AMPure cleanup
67. Remove and retain 10 μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Single Primer PCR amplification
68. Setup PCR master mix:

Component                                    Volume
2x LongAmp PCR buffer      12.5 µL
SISPA-B primer 10 uM            5 µL
Nuclease-free water              5 µL

 

SECTION: Single Primer PCR amplification
69. Program the following PCR cycles into the thermal cycler.

Step                                                   Temperature                 Time                        Cycles
Inital Denaturation                         95C                    3 min                        1                
Denaturation                                  95C                      15 s                       20
Annealing                                       50C                      30 s                       20
Extension                                       65C                      50 s                       20
Final Extension                              65C                     5 min                        1
SECTION: AMPure cleanup 2
70. Prepare the AMPure XP beads for use; resuspend by vortexing.
SECTION: AMPure cleanup 2
71. Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: AMPure cleanup 2
72. Add 25 μl of resuspended AMPure XP beads to the PCR product and mix by pipetting up and down.
SECTION: AMPure cleanup 2
73. Incubate for 5 minutes at room temperature.
SECTION: AMPure cleanup 2
74. Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
SECTION: AMPure cleanup 2
75. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: AMPure cleanup 2
76. Keep on magnet, wash beads with 200 μl of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard.
SECTION: AMPure cleanup 2
77. Repeat the previous step.
SECTION: AMPure cleanup 2
78. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 3 min, but do not dry the pellet to the point of cracking.
SECTION: AMPure cleanup 2
79. Remove the tube from the magnetic rack and resuspend pellet in 24 μl nuclease-free water. Incubate for 2 minutes at room temperature
SECTION: AMPure cleanup 2
80. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: AMPure cleanup 2
81. Remove and retain 24 μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
 

SECTION: Native barcoding and sequencing by ligation
82. Refer to the nanopore protocol.
SECTION: Reverse Transcription
49. Prepare RT mastermix:

Component                      Volume
5x SSIV Buffer                 2 µL
DTT 100 mM                  0.5 µL
RNase Inhibitor              0.5 µL
Reverse Transcriptase  0.5 µL

SECTION: Reverse Transcription
50. Add 3.5ul of RT master mix to annealed RNA.
SECTION: Reverse Transcription
51. Incubate at 42C for 10 min and then at 80C for 10 min.
SECTION: FastSelect rRNA removal
27. Bring FastSelect -rRNA HMR, FastSelect 5S/16S/23S, FastSelect FH Buffer, and Nuclease-free Water to room temperature.
SECTION: FastSelect rRNA removal
28. For FastSelect 5S/16S/23S, incubate the tube at 37°C for 5 min, and then vortex to dissolve the precipitate.
SECTION: FastSelect rRNA removal
29. Mix all other reagents vigorously by vortexing, and then briefly centrifuge.
SECTION: FastSelect rRNA removal
30. Prepare depletion master mix on ice:

Component                           Volume
FastSelect FH Buffer     1.5 µL
FastSelect -rRNA HMR   1 µL
FastSelect 5S/16S/23S   1 µL
Nuclease-free Water      1.5 µL

SECTION: FastSelect rRNA removal
31. Add 5 ul of master mix to template RNA.
SECTION: FastSelect rRNA removal
32. Incubation reactions as follows:

1. 2 min at 75 °C
2. 2 min at 70 °C
3. 2 min at65 °C
4. 2 min at60 °C
5. 2 min at 55 °C
6. 2 min at37 °C
7. 2 min at 25 °C
8. Hold at 4°C
SECTION: FastSelect rRNA removal
33. Add 19.5 μl QIAseq Beads (prewarmed to room temperature and mixed by vortexing) to the 15 μl reaction. 

Mix thoroughly by vortexing, and then incubate for 5 min at room temperature.
SECTION: FastSelect rRNA removal
34. Centrifuge in a table-top centrifuge until the beads are completely pelleted (~2 min).
SECTION: FastSelect rRNA removal
35. Place the tubes/plate on a magnetic rack for 2 min. Once the solution has cleared, with the beads still on the magnetic stand, carefully remove and discard the supernatant.
 

SECTION: FastSelect rRNA removal
36. Add 15 μl of Nuclease-free Water and 19.5 μl of QIAseq Bead Binding Buffer (prewarmed to room temperature and mixed by vortexing). Mix vigorously by vortexing, and then incubate for 5 min at room temperature.
SECTION: FastSelect rRNA removal
37. Centrifuge in a table-top centrifuge until the beads are completely pelleted (~2 min).
SECTION: FastSelect rRNA removal
38. Place the tubes/plate on a magnetic rack for 2 min. Once the solution has cleared, with the beads still on the magnetic stand, carefully remove and discard the supernatant.
SECTION: FastSelect rRNA removal
39. With the beads still on the magnetic stand, add 200 μl of 80% ethanol. Wait for 30 s.

Carefully remove and discard the wash.

 

SECTION: FastSelect rRNA removal
40. Repeat the ethanol wash.

 

SECTION: FastSelect rRNA removal
41. With the beads still on the magnetic stand, air-dry at room temperature for 6–10 min until all liquid has evaporated but without overdrying the beads.
 

SECTION: FastSelect rRNA removal
42. Remove the beads from the magnetic stand, and elute the RNA from the beads by adding 12 μl Nuclease-free Water. Mix well by pipetting or vortexing, and then allow the tubes to sit at room temperature for 2 min to appropriately hydrate the beads.
SECTION: FastSelect rRNA removal
43. Return the tubes/plate to the magnetic rack until the solution has cleared.
SECTION: FastSelect rRNA removal
44. Transfer 10 μl of the supernatant to nuclease-free tube.
 

SECTION: Sample Preprocessing
1. Refer to sample preprocessing protocol for appropriate sample type.