TITLE: Sequence-Independent, Single-Primer Amplification of RNA viruses
AUTHORS: Gage Moreno, David O'connor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the methods to perform unbiased direct metagenomic sequencing of nucleic acid extracts from cell-free fluids. This protocol can be adapted to be run on Illumina and Nanopore sequencing platforms. </div><div class = "text-block">The protocol is based off of the work from Kafetzopoulou et al. (PMID: 30563591). Liana has provided the lab with detailed protocols, and has worked with us extensively on optimizing and getting protocols running efficiently. Please note that this protocol has been updated to use SuperScript IV with it’s optimal temperature which has been reflected in Lewandowski et al. (DOI: https://doi.org/10.1128/JCM.00963-19)</div><div class = "text-block">Notes:</div><div class = "text-block">* This protocol has been used to sequence influenza direction from respiratory clinical samples (DOI: https://doi.org/10.1128/JCM.00963-19).</div><div class = "text-block">* A team from China published last week on a 2019-nCoV familial cluster using the SISPA protocol as for coronavirus whole genome sequencing (DOI: https://doi.org/10.1016/S0140-6736(20)30154-9)</div><div class = "text-block">SISPA-Primer A - 5'-GTT TCC CAC TGG AGG ATA-(N9)-3'</div><div class = "text-block">SISPA-Primer B - 5′-GTT TCC CAC TGG AGG ATA-3′</div></div>

[STEPS]
?. [Nucleic Acid Extraction]
Add 280µL cell-free liquid to 0.22µm centrifuge filter. Note: (add in 2x what you want to get out)
?. [Nucleic Acid Extraction]
Centrigue at 14,000 RPM for 5 minutes
Centrifuge: 14000 33, 5 min
?. [Nucleic Acid Extraction]
Transfer 140µL of filtered sample to a new tube.
?. [Nucleic Acid Extraction]
Prepare Buffer AVL and linear polyacrylamide mix. * For one sample, 560µL Buffer AVL + 5.6µL linear polyacrylamide Reagent Volume (µL)
?. [Nucleic Acid Extraction]
Pipet 560μL prepared Buffer AVL containing linear polyacrylamide into a 1.5 ml microcentrifuge tube. * Note: If the sample volume is larger than 140 μl, increase the amount of Buffer AVL–linear polyacrylamide proportionally (e.g., a 280 μl sample will require 1120 μl Buffer AVL–linear polyacrylamide) and use a larger tube.
?. [Nucleic Acid Extraction]
Add 140 μl plasma, serum, urine, cell-culture supernatant or cell-free body fluid to the Buffer AVL–LPA in the microcentrifuge tubes. Mix by pulse-vortexing for 15 s. * Note: To ensure efficient lysis, it is essential that the sample is mixed thoroughly with Buffer AVL to yield a homogeneous solution. Frozen samples that have only been thawed once can also be used.
?. [Nucleic Acid Extraction]
Incubate at room temperature (15–25°C) for 10 min. * Note: Viral particle lysis is complete after lysis for 10 min at room temperature. Longer incubation times have no effect on the yield or quality of the purified RNA.
?. [Nucleic Acid Extraction]
Briefly centrifuge the tube to remove drops from the inside of the lid.
?. [Nucleic Acid Extraction]
Add 560 μl ethanol (96–100%) to each sample, and mix by pulse-vortexing for 15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid. * Note: Use only ethanol, since other alcohols may result in reduced RNA yield and purity. Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. If the sample volume is greater than 140 μl, increase the amount of ethanol proportionally (e.g., a 280 μl sample will require 1120 μl ethanol). To ensure efficient binding, it is essential that the sample is mixed thoroughly with the ethanol to yield a homogeneous solution.
?. [Nucleic Acid Extraction]
Carefully apply 630 μl of the solution from step 8 to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube, and discard the tube containing the filtrate. * Note: Close each spin column to avoid cross-contamination during centrifugation. * Note: Centrifugation is performed at 6000 x g (8000 rpm) to limit microcentrifuge noise. Centrifugation at full speed will not affect the yield or purity of the viral RNA. If the solution has not completely passed through the membrane, centrifuge again at a higher speed until all of the solution has passed through.
Centrifuge: 8000 33, 1 min
?. [Nucleic Acid Extraction]
Carefully open the QIAamp Mini column, and repeat step 9. If the sample volume was greater than 140 μl, repeat this step until all of the lysate has been loaded onto the spin column.
?. [Nucleic Acid Extraction]
Carefully open the QIAamp Mini column, and add 500 μl Buffer AW2. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Continue directly with step 14, or to eliminate possible Buffer AW2 carryover, perform step 13 and then continue. * Note: Residual Buffer AW2 in the eluate may cause problems in downstream applications. Some centrifuge rotors may vibrate upon deceleration, resulting in flow- through, containing Buffer AW2, contacting the QIAamp Mini column.
Centrifuge: 14000 33, 3 min
?. [Nucleic Acid Extraction]
Recommended: Place the QIAamp Mini column in a new 2 ml collection tube (not provided), and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.
Centrifuge: 14000 33, 1 min
?. [Nucleic Acid Extraction]
Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube (not provided). Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini column and add 30 μl DNase/RNase Free Water equilibrated to room temperature. Close the cap, and incubate at room temperature for 1 min.800
?. [Nucleic Acid Extraction]
Centrifuge at 6000 x g (8000 rpm) for 1 min.
Centrifuge: 8000 33, 1 min
?. [Nucleic Acid Extraction]
Transfer 30µL of eluted nucleic acid to a new microcentrifuge tube.
?. [DNase Treatment]
Create a master mix of TURBO DNase Mix:Add  1 μL TURBO DNase (2 U) (for up to 10 μg RNA in a 50 μL reaction), 5µL TURBO DNase Buffer, and 14µL of H2O per sample.
?. [DNase Treatment]
Add 20µL of TURBO DNase Mix to 30µL of sample.
?. [DNase Treatment]
Incubate at 37°C for 30 minutes.
37 °C
?. [Zymo Clean-up and Concentrator]
Add 2 volumes RNA Binding buffer to each sample (ex. 100µL buffer + 50 µL sample)
?. [Zymo Clean-up and Concentrator]
Add an equal amount of 100% ethanol and mix. (ex. 150µL ethanol)
?. [Zymo Clean-up and Concentrator]
Transfer the sample to the Zymo-Spin IC Column in a Collection Tube and centrifuge for 30 seconds. Discard the flow-through * For samples >800 µL, Zymo-Spin columns can be reloaded
Centrifuge: 14000 33, 30 s
?. [Zymo Clean-up and Concentrator]
Add 400 µL RNA Prep Buffer to the column and centrifuge at 10,000g– 16,000g for 30 seconds. Discard flow-through.
Centrifuge: 14000 33, 30 s
?. [Zymo Clean-up and Concentrator]
Add 700 µL RNA Wash Buffer to the column and centrifuge at 10,000g– 16,000g for 30 seconds. Discard flow-through.
Centrifuge: 14000 33, 30 s
?. [Zymo Clean-up and Concentrator]
Centrifuge at 10,000g– 16,000g for 2 minutes to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase free tube (Not provided in RNA Clean & Concentrator-5 kit).
Centrifuge: 14000 33, 2 min
?. [Zymo Clean-up and Concentrator]
Add 8-10 µL Nuclease-Free water directly to the column matrix and centrifuge at 10,000g– 16,000g for 30 seconds. The eluted RNA can be used immediately or stored at -70°C.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
Make a working stock of your primer A. Stock: 100pmol/1µL. Add 4µL of stock + 6µL H2O. You now have a 10µL of a 40pmol/µL working stock.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
Add 1 µL Primer A working stock to 4 µL extracted RNA. Heat to 65°C for 5 minutes in a thermocycler and let cool at 4°C for 5 minutes.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of 2 μL 5X RT buffer, 1 μL 10 mM dNTP, 1 μL water, 0.5 μL 0.1M DTT, and 0.5 μL SSIV RT.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
Add 5 µL master mix to reaction. Incubate at 50°C for 10 minutes.
50 °C
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of 1 μL 5X Sequenase buffer, 3.8 μL water, and 0.15 μL Sequenase.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
After 10 minute incubation, Add 5 µL of Sequenase Mix #1 to the reaction.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
Incubate at  37°C for 8 min.
37 °C
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
While reaction is on the thermocycler, make a master mix for 1 reaction (scale up as needed) consisting of 0.45 μL Sequenase dilution buffer, and 0.15 μL Sequenase.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
After 8 min incubation, add 0.6µL of Sequenase Mix #2 to the reaction.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
Incubate at  37°C for 8 min.
37 °C
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
After 8 min incubation, add 1µL of RNase H to each sample.
?. [SISPA A: Reverse Transcription and 2nd strand cDNA synthesis Primer A Addition]
Incubate reaction at  37°C for 20 min.* Round A is now complete and samples can be stored at -20°C
37 °C
?. [SISPA B: PCR Amplification of Randomly Primed cDNA cDNA Amplification]
Make a master mix for 1 reaction (scale up as needed) consisting of 5 µL AccuTaq LA 10x Buffer, 2.5 µL dNTP mix, 1µL DMSO, 0.5 µL AccuTaq LA DNA Polymerase, 35 µL nuclease free water, and 1 µL Primer B. *Do not dilute Primer B
?. [SISPA B: PCR Amplification of Randomly Primed cDNA cDNA Amplification]
Add 5 µL of product from SISPA Round A to 45 µL master mix.
?. [SISPA B: PCR Amplification of Randomly Primed cDNA cDNA Amplification]
Run PCR with the following conditions 98°C for 30s, followed by 30 cycles of 94°C for 15 s, 50°C for 20 s, and 68°C for 2 min, a final step of 68°C for 10 min, and then a 4°C hold until you’re ready for the cleanup. * Can freeze in the -20°C until ready
?. [Bead Clean-up]
Amplified cDNA was purified using a 1:1 ratio of AMPure XP beads. Add 50µL of resupsended AMPure XP beads to 50µL SISPA product.
?. [Bead Clean-up]
Incubate at RT for 10 minutes
0 Room temperature
?. [Bead Clean-up]
Spin down briefly
?. [Bead Clean-up]
Place on magnet. Remove and discard supernatant once solution turns clear. Be sure not to disturb beads.
?. [Bead Clean-up]
Wash with 200µL of 70% EtOH.
?. [Bead Clean-up]
Remove EtOH without disturbing beads.
?. [Bead Clean-up]
Repeat wash. Remove EtOH without disturbing beads.
?. [Bead Clean-up]
Remove samples from magnetic rack and spin down briefly.
?. [Bead Clean-up]
Remove residual ethanol
?. [Bead Clean-up]
Let air dry briefly - Do not overdry bead pellet
?. [Bead Clean-up]
Resuspend sample in 50µL of H2O.
?. [Bead Clean-up]
Incubate samples at RT for 5 minutes.
0 Room temperature
?. [Bead Clean-up]
Transfer tubes to magnetic rack. Transfer 48µL of eluted product to a new tube.
?. [Bead Clean-up]
Continue onto library prep protocol of your choice.