TITLE: Generation of SARS-COV-2 RNA transcript standards for qRT-PCR detection assays
AUTHORS: Chantal Vogels, Joseph Fauver, Isabel Ott, Nathan Grubaugh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The protocol describes how to generate high-quality single-stranded RNA transcript standards (starting from a virus RNA stock) targeting the nsp10, RdRp, nsp14, envelope (E), and nucleocapsid (N) coding regions for use with </span><a href="http://ivdc.chinacdc.cn/kyjz/202001/t20200121_211337.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">China CDC</span></a><span>, </span><a href="https://www.who.int/docs/default-source/coronaviruse/peiris-protocol-16-1-20.pdf?sfvrsn=af1aac73_4" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Hong Kong University (HKU)</span></a><span>, </span><a href="https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.3.2000045" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Corman et al. (Berlin)</span></a><span>, and </span><a href="https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">US CDC</span></a><span> SARS-CoV-2 </span><a href="https://docs.google.com/spreadsheets/d/1kWLdaqWZZYt383EMSf-osyXGdeedrRF6YeBuOBin6Ok/edit#gid=0" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">primer and probe sets for qRT-PCR</span></a><span>. (Sequences for transcripts generated, along with their corresponding assays, are provided under 'Guidelines.')</span></div></div>

[STEPS]
?. [Preparation of cDNA]
Isolate viral RNA using Omega Viral DNA/RNA kit, Trizol, or equivalent.
?. [Preparation of cDNA]
Many different cDNA synthesis kits can be used, but choose something that is relatively high-fidelity. The current protocol uses SuperScript IV VILO Master Mix because the enzyme has low error rates and the protocol is fast and easy.  AB1ComponentVolume in 20 µL reaction2SSIV VILO Master Mix4 µL3Nuclease-free water11 µL4Virus RNA5 µL
AB1ComponentVolume in 20 µL reaction2SSIV VILO Master Mix4 µL3Nuclease-free water11 µL4Virus RNA5 µL
?. [Preparation of cDNA]
Run the following cycles on a thermocycler:  AB1TemperatureTime225°C10 minutes350°C10 minutes485°C5 minutes54°C∞
AB1TemperatureTime225°C10 minutes350°C10 minutes485°C5 minutes54°C∞
?. [Preparation of cDNA]
Store samples at   or  until ready for PCR.
[(same day)]
[(up to a week)]
?. [PCR]
Prepare the following primers at a concentration of 10 µM, depending on which assay is needed. The nucleocapsid (N) transcript is for the US CDC-N1, N2, and N3 (N3 is not currently in use), HKU-N, and China CDC-N assays. The envelope (E) transcript is for use with the Berlin-E assay, nsp14 transcript is for use with the HKU-ORF1 assay, the RdRp transcript is for use with the Berlin RdRp assay (low sensitivity), and the nsp10 transcript is for use with the China CDC-ORF1 assay. See here for assay primer sequences and references.  ABCDE1TargetPrimerSequencePositionAmplicon size2NN-Std-T7-FwdTAATACGACTCACTATAGGGGAATTGTGCGTGGATGAGGC2806813633N-Std-RevTGTCTCTGCGGTAAGGCTTG294114EE-Std-T7-FwdTAATACGACTCACTATAGGGGCGTGCCTTTGTAAGCACAA262078085E-Std-RevGGCAGGTCCTTGATGTCACA270166nsp14nsp14-Std-T7-FwdTAATACGACTCACTATAGGGTAGTGCTAAACCACCGCCTG184478487nsp14-Std-RevAACTGCCACCATCACAACCA192758RdRpRdRp-Std-T7-FwdTAATACGACTCACTATAGGGAATAGAGCTCGCACCGTAGC150948839RdRp-Std-RevCATCTACAAAACAGCCGGCC1595710nsp10nsp10-Std-T7-FwdTAATACGACTCACTATAGGGGTGGGGGACAACCAATCACT1312270411nsp10-Std-RevAGACGAGGTCTGCCATTGTG13806
ABCDE1TargetPrimerSequencePositionAmplicon size2NN-Std-T7-FwdTAATACGACTCACTATAGGGGAATTGTGCGTGGATGAGGC2806813633N-Std-RevTGTCTCTGCGGTAAGGCTTG294114EE-Std-T7-FwdTAATACGACTCACTATAGGGGCGTGCCTTTGTAAGCACAA262078085E-Std-RevGGCAGGTCCTTGATGTCACA270166nsp14nsp14-Std-T7-FwdTAATACGACTCACTATAGGGTAGTGCTAAACCACCGCCTG184478487nsp14-Std-RevAACTGCCACCATCACAACCA192758RdRpRdRp-Std-T7-FwdTAATACGACTCACTATAGGGAATAGAGCTCGCACCGTAGC150948839RdRp-Std-RevCATCTACAAAACAGCCGGCC1595710nsp10nsp10-Std-T7-FwdTAATACGACTCACTATAGGGGTGGGGGACAACCAATCACT1312270411nsp10-Std-RevAGACGAGGTCTGCCATTGTG13806
?. [PCR]
Prepare the following PCR reaction for each sample:  AB1ComponentVolume in 25 µL reaction2Q5 2x Master Mix12.5 µL310 µM Primer F-T71.25 µL410 µM Primer R1.25 µL5Nuclease-free water8 µL6cDNA2 µL
AB1ComponentVolume in 25 µL reaction2Q5 2x Master Mix12.5 µL310 µM Primer F-T71.25 µL410 µM Primer R1.25 µL5Nuclease-free water8 µL6cDNA2 µL
?. [PCR]
Run the following cycles on a thermocycler:  AB1TemperatureTime298°C30 seconds398°C10 seconds455°C30 seconds572°C90 seconds6Repeat steps 2-4 for a total of 35 cycles772°C2 minutes84°C∞
AB1TemperatureTime298°C30 seconds398°C10 seconds455°C30 seconds572°C90 seconds6Repeat steps 2-4 for a total of 35 cycles772°C2 minutes84°C∞
?. [Post-PCR cleanup]
Allow Mag-Bind TotalPure NGS beads to equilibrate to room temperature, vortex until homogenous.
?. [Post-PCR cleanup]
Bring PCR product volume up to 25 µL with water (if not at volume already).
?. [Post-PCR cleanup]
Add 20 µL of beads to 25 µL of PCR product, mix well, and incubate at room temperature for 10 minutes.
If your lab has a KingFisher extractor to use for post-PCR cleanup steps, our automated protocols are available here: https://github.com/grubaughlab/Kingfisher_protocols (use ‘purification.bdz’ for this cleanup)
?. [Post-PCR cleanup]
Place tubes on a magnetic stand and incubate until solution appears clear.
?. [Post-PCR cleanup]
Discard supernatant without disturbing the beads.
?. [Post-PCR cleanup]
While tubes are on the magnet, add 200 µL of 80% ethanol, incubate for 30 seconds, and discard the ethanol wash.
?. [Post-PCR cleanup]
Repeat previous 80% ethanol wash and remove as much ethanol as possible.
?. [Post-PCR cleanup]
Leave tubes on magnet and air dry for 5 minutes.
?. [Post-PCR cleanup]
Remove tubes from magnet and add 20 µL of nuclease-free water. Mix well by pipetting.
?. [Post-PCR cleanup]
Place tubes on magnet stand. When solution appears clear, remove supernatant without disturbing the beads and place into new tubes.
?. [Intermediate quantification]
Quantify the DNA concentration using the Qubit High Sensitivity DNA kit (or equivalent) from 1 µL of each product, following manufacturer's protocol.
?. [Intermediate quantification]
Check DNA fragment distributions of the samples using the BioAnalyzer DNA 1000 kit, following manufacturer's protocol.
?. [Reverse Transcription (Megascript T7 kit)]
Important: thaw reagents on ice, but keep the 10X reaction buffer at room temperature while assembling the reaction. Add the 10X reaction buffer after water and NTPs have been added to the tube.
?. [Reverse Transcription (Megascript T7 kit)]
Mix equal volumes (2 µL/sample) of the four ribonucleotide solutions together and add 8 µL of the mixture for each sample.
?. [Reverse Transcription (Megascript T7 kit)]
Use 2 µL of pTRI-Xef in a standard reaction as a transcription control. This should yield a transcript of 1.89 kb.  AB1ComponentVolume in 20 µL reaction2Nuclease-free waterAdd to 20 µL3NTP mixture (ATP + CTP + GTP + UTP)8 µL410X reaction buffer2 µL5Purified PCR product0.1-0.2 µg PCR product6Enzyme mix2 µL
AB1ComponentVolume in 20 µL reaction2Nuclease-free waterAdd to 20 µL3NTP mixture (ATP + CTP + GTP + UTP)8 µL410X reaction buffer2 µL5Purified PCR product0.1-0.2 µg PCR product6Enzyme mix2 µL
?. [Reverse Transcription (Megascript T7 kit)]
Mix by pipetting up and down and microfuge briefly to collect reaction mixture at the bottom of the tube.
?. [Reverse Transcription (Megascript T7 kit)]
Incubate at .
[for 4 hours]
?. [Reverse Transcription (Megascript T7 kit)]
Add 1 µL of TURBO DNase, mix well and incubate .
[for 15 minutes]
?. [RNA product purification]
Add the following components to a 1.5 mL tube.  AB1ComponentVolume in 81 µL reaction2Nuclease-free water30 µL3Lithium Chloride30 µL4T7 transcription product21 µL
AB1ComponentVolume in 81 µL reaction2Nuclease-free water30 µL3Lithium Chloride30 µL4T7 transcription product21 µL
?. [RNA product purification]
Mix and chill at .
[for >30 minutes]
?. [RNA product purification]
Centrifuge at  at maximum speed to pellet the RNA.
[for 15 minutes]
?. [RNA product purification]
Carefully remove supernatant. Wash pellet with 1 mL of 70% ethanol and re-centrifuge at  at maximum speed to maximize removal of unincorporated nucleotides.
[for 15 minutes]
?. [RNA product purification]
Carefully remove the ethanol and resuspend in 20 µL of nuclease-free water.
?. [Final quantification]
Quantify the RNA concentration using the Qubit High Sensitivity RNA kit from 1 µL of each product, following manufacturer's protocol.
?. [Final quantification]
Check RNA fragment distributions of the samples using the BioAnalyzer RNA 6000 pico kit,  following manufacturer's protocol.
?. [Final quantification]
Use the following RNA copy number calculator to calculate genome equivalents per uL and prepare standards.