TITLE: Preparation of dsRNA viruses for MinION - CLM
AUTHORS: Alexander H Wilcox, Erik Delwart, Samuel L Díaz Muñoz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Updated protocol by CLM</div></div>

[STEPS]
?. Viral lysates should be passed through a 0.22μm filter to remove host debris, then treated with nucleases to degrade extracapsular nucleic acids.  To 1ml filtrate:25μl DNAse I, 50μl RNAse A/T1107.5 DNAse I BufferScale down for concentrated lysates, which should be in the range of 50-500uL.Incubate for 1 hour 30 minutes at 37ºC.Inactivate nucleases at 65ºC for 10 minutes and proceed to RNA extraction.
?. RNA should be extracted using a commercially available kit.  We used an RNeasy Mini Kit (Qiagen).  Kits that require carrier RNA are not recommended because this will interfere with sequencing.Follow manufacturer instructions with the following modifications:1. Pass all lysate through column at the first step (i.e. add buffer + lysate mix multiple times until lysate is used up)2. Elute with 45uL of Elution Buffer. Let column sit at 25ºC for 5 minutes before spinning.3. Split into 20uL aliquots. RNA may be frozen at -80ºC for future processing or may move on to next step.
?. Quantify the RNA using Qubit RNA High Sensitivity Kit (HS).  Note that low viral titres and/or small viral genomes may result in the amount of RNA being below the Qubit's limit of detection.
?. Add 50% DMSO (v/v) to RNA sample, and incubate for 1 hour 30 minutes at 65ºC.
?. Purify RNA using a column cleanup kit (we recommend Qiagen's RNeasy MinElute Cleanup Kit) or your preferred other method (e.g. ethanol precipitation, TRIzol purification). This step removes DMSO from sample.If using RNeasy MinElute Cleanup Kit: Adjust sample volume to 100 uL using RNade free water (ex: if sample volume = 20 uL RNA + 10 uL DMSO, add 70 uL RNase free water)Centrifuge ‘full speed’ == 21000 rcf or highest speed on centrifugeStore at -80 ºC or place on ice and proceed to first strand synthesis
?. First strand synthesis - Superscript IV (in BSC):Calculate reagent volumes for MM:ReagentVol for 1 rxndNTPs1 uLRandom hexamers1 uLRNase free water4 uLReagentVol for 1 rxn5x SSIV buffer4 uL100 mM DTT1 uLRNase out *1 uLSSIV RT1 uL1. Mix appropriate volume (# samples+ 1) of 1st step mastermix and add 6 uL of MM to each tube            Keep MM tube on ice            Start with RNase free water2. Add 7 uL of each RNA eluate to a tube strip and label + record scheme in notebook            keep on cooling tray, do not allow to freeze            Total vol of each tube is 13 uL            Minifuge3. Place on thermal cycle, start incubation at 65ºC for 5 minutes. At end of 5 min, place on ice for at least 1 min4. During 5 min incubation, mix appropriate volume (# samples+ 1) of 2nd step mastermix.            Get SSIV RT on cold block from freezer            Keep MM tube on ice            Mix SSIV buffer            Add SSIV RT last (pipette slowly)5. Add 7 uL (vol for 1 rxn) of MM to each sample on the tube strip             Keep tube strip on cooling tray            Total vol of each tube is now 20 uL            Minifuge6. Place back on thermal cycler and proceed thru the following incubation steps:             10 min at 23 ºC            10 min at 52.5 ºC            10 min at 80 ºC7. Store at 4 ºC or place reactions on ice and proceed to second strand synthesis.
ReagentVol for 1 rxndNTPs1 uLRandom hexamers1 uLRNase free water4 uL
ReagentVol for 1 rxn5x SSIV buffer4 uL100 mM DTT1 uLRNase out *1 uLSSIV RT1 uL
?. Second strand synthesis. Calculate reagent volumes for MM: ReagentVol for 1 rxndNTPs1 uLE. Coli DNA ligase0.5 uLDNA polymerase I2 uLRNase H0.5 uL5x second strand synthesis buffer (OR 10x second strand synthesis buffer)8uL(4uL)Nuclease free water8 uL (use 12 uL if used 10x buffer) 1. Mix appropriate volume (# samples+ 1) of second strand mastermix.            Keep MM tube on ice            Keep enzymes on cold block            Start with nuclease free water            Add enzymes last2. Add 20 uL (vol for 1 rxn) of MM to each sample on the tube strip             Keep tube strip on cooling tray            Total vol of each tube is now 20 uL            Minifuge3. Place on thermal cycler and begin 5 hour incubation at 16 ºC4. Store at 4 ºC or proceed to cleanup
ReagentVol for 1 rxndNTPs1 uLE. Coli DNA ligase0.5 uLDNA polymerase I2 uLRNase H0.5 uL5x second strand synthesis buffer (OR 10x second strand synthesis buffer)8uL(4uL)Nuclease free water8 uL (use 12 uL if used 10x buffer)
?. Purify DNA using your preferred method (we used a Macharey-Nagal Nucleospin Gel and PCR Clean-up kit, per manufacturer instructions). Quantify DNA using Qubit.
?. Prepare DNA libraries for sequencing using your chosen platform and library preparation kit (we used Nextera XT, we will modify this protocol for Oxford Nanopore Native Barcode sequencing).