TITLE: Viral metagenomics using SMART-9n amplification and nanopore sequencing
AUTHORS: Ingra Claro Morales, Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a SMART-Seq (Switch Mechanism at the 5′ End of RNA Templates) protocol developed to use random 9n priming and be compatible with ONT RLB rapid, barcoded adapters available from the SQK-RPB004 kit. We have generated cDNA reads in excess of 10 kb from Zika viral reference material with this protocol.</div></div>

[STEPS]
?. [Centrifugal filtration]
Transfer up to  sample directly onto a Ultrafree-MC Centrifugal Filter column
500 µl
?. [Centrifugal filtration]
Spin at  for
Centrifuge: 5000 34
?. [Centrifugal filtration]
Recover filtrate into Eppendorf tube
1.5 ml
?. [Centrifugal filtration]
Remove basket and discard
?. [Centrifugal filtration]
Close lid and place on ice
?. [Viral RNA extraction]
In a Eppendorf tube combine sample, DNA/RNA Shield (2x concentrate) and mix well by pipetting
2 ml
200 µl
200 µl
?. [Viral RNA extraction]
Add  Viral RNA Buffer and mix well by pipetting
800 µl
?. [Viral RNA extraction]
Load  onto a column in a collection tube and spin at  for discard flow through, place in a new collection tube
600 µl
Centrifuge: 10000 34
?. [Viral RNA extraction]
Add  Viral wash buffer and spin at  for , discard flow through
500 µl
Centrifuge: 10000 34
?. [Viral RNA extraction]
Add  100% ethanol and spin at  for , discard flow through, place in a clean  tube
500 µl
Centrifuge: 10000 34
1.5 ml
?. [Viral RNA extraction]
Add  of DNA/RNA-Free Water and incubate at RT for
15 µl
?. [Viral RNA extraction]
Spin at  for
Centrifuge: 10000 34
?. [DNase Treatment]
Set heat block to
37 °C
?. [DNase Treatment]
Set up the following reaction: Component                                VolumeRNA                                    	    10X TURBO DNase Buffer 	    TURBO DNase                   	    Total
44 µl
5 µl
1 µl
50 µl
?. [DNase Treatment]
Incubate at  for
37 °C
?. [DNase cleanup]
Add  RNA Binding Buffer and mix by vortexing  and spin down
100 µl
?. [DNase cleanup]
Add 100% ethanol and mix by vortexing for  and spin down
150 µl
?. [DNase cleanup]
Transfer  to a Zymo-Spin IC column in a collection tube and spin at  for discard flow through
300 µl
2 ml
Centrifuge: 6000 34
?. [DNase cleanup]
Add  RNA Prep Buffer and spin at  for , discard flow through
400 µl
Centrifuge: 6000 34
?. [DNase cleanup]
Add  RNA Wash Buffer and spin at  for , discard flow through and place in a new  Eppendorf tube
700 µl
Centrifuge: 6000 34
1.5 ml
?. [DNase cleanup]
Add DNase/RNase Free Water and incubate at RT for
10 µl
?. [DNase cleanup]
Spin at  for
Centrifuge: 6000 34
?. [DNase cleanup]
Label as ‘Viral RNA’ and place on ice
?. [Viral DNA extraction]
Set heat block to
55 °C
?. [Viral DNA extraction]
In a  Eppendorf tube combine  sample,  DNA/RNA Shield (2X concentrate), Proteinase K and mix well by pipetting
2 ml
200 µl
200 µl
20 µl
?. [Viral DNA extraction]
Incubate at  for
55 °C
?. [Viral DNA extraction]
Set heat block to
60 °C
?. [Viral DNA extraction]
Add binding buffer and mix well
1.2 ml
?. [Viral DNA extraction]
Load  onto a Zymo-Spin IIC-Z column in a collection tube and spin at  for , discard the discard flow-through and reload as many times as required
800 µl
Centrifuge: 8000 34
?. [Viral DNA extraction]
Transfer to to a new collection tube add  DNA Wash Buffer 1 and spin at  for , discard flow-through
400 µl
Centrifuge: 8000 34
?. [Viral DNA extraction]
Add DNA Wash Buffer 2 and spin at  for .
700 µl
Centrifuge: 8000 34
?. [Viral DNA extraction]
Add  DNA Wash Buffer 2 and spin at  for .
200 µl
Centrifuge: 8000 34
?. [Viral DNA extraction]
Transfer to a new  Eppendorf tube and  DNA/RNA Free Water preheated to  to the column, incubate at RT for .
1.5 ml
50 µl
60 °C
?. [Viral DNA extraction]
Spin at  for .
Centrifuge: 8000 34
?. [SMART-9n amplification]
Combine the following in a 0.2ml 8-strip tube.Component                            VolumeRLB RT 9N (2 uM)                dNTPs (10 mM ea.)		Template RNA			Total
1 µl
1 µl
10 µl
12 µl
?. [SMART-9n amplification]
Mix and spin down.
?. [SMART-9n amplification]
Incubate at  for  then snap cool on ice.
65 °C
?. [SMART-9n amplification]
Make up the following master mix and add to the  annealed RNA:Component                                    VolumeSSIV buffer (5x)			        DTT (100 mM)				RNase OUT				        SS IV RTase (200 U/ul)		RLB TSO (2 uM)			        Total
8 µl
12 µl
4 µl
1 µl
1 µl
1 µl
1 µl
20 µl
?. [SMART-9n amplification]
Start the following program on a thermocycler:for for
42 °C
70 °C
?. [SMART-9n amplification]
Set up the following reaction to amplify cDNA in a new 0.2ml 8-strip tube:Component                                        VolumeLongAmp Taq 2X master mix	      RLB (01-12)				              NFW					              cDNA                                                    Total
25 µl
0.5 µl
19.5 µl
5 µl
50 µl
?. [SMART-9n amplification]
Start the following program on the thermal cycler:  Step                        Temperature         Time                        CyclesHeat Activation              1Denaturation                  26Annealing                       26Extension                        26Final extension              1Hold                                      Indefinite                    1
95 °C
95 °C
56 °C
65 °C
65 °C
4 °C
?. [PCR clean-up]
Clean up products with 1x Ampure XP and elute in 30 ul EB
?. [QC]
Quantify the PCR products using the Quantus Fluorometer using the ONE dsDNA assay.
?. [Rapid adapter attachment]
Pool all barcoded products to a total of  in  of  Tris-HCl pH 8.0 with  NaCl
10 µl
?. [Rapid adapter attachment]
Add  RAP adapter and mix by pipetting, incubate at RT
1 µl
?. [MinION sequencing]
Prime the flowcell and load sequencing library onto the flowcell.