TITLE: NEBNext® Varskip Short ARTIC SARS-CoV-2 RT-PCR Module E7626
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module, NEB #E7626S/L 24/96 reactions. </div><br/><div class = "text-block">cDNA Synthesis and Targeted cDNA Amplification with NEBNext VarSkip Short Primer Mixes: </div><div class = "text-block">This protocol follows an alternate variant-tolerant approach for targeting SARS-CoV-2 by utilizing NEBNext VarSkip Short SARS-CoV-2 Primer Mixes. The NEBNext VarSkip Short SARS-CoV-2 Primer mixes cannot be added to the same cDNA amplification reaction as the NEBNext ARTIC SARS-CoV-2 Primer Mixes. If downstream applications include sequencing, performing RNA input normalization prior to cDNA synthesis and targeted amplification promotes more even distribution of reads across sequencing libraries.</div><br/><div class = "text-block"><span>For other NEBNext® ARTIC SARS-CoV-2 protocols, please see the </span><a href="https://www.protocols.io/edit/nebnext-artic-protocols-collection-bw2apgae" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEBNext ARTIC Protocols Collection</span></a><span>. </span></div><br/><div class = "text-block"><span>To obtain instructions for using NEBNext ARTIC SARS-CoV-2 Primer Mix and the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module please see the </span><a href="https://www.neb.com/products/e7626-nebnext-artic-sars-cov-2-rt-pcr-module%23Protocols,%20Manuals%20&%20Usage_Manuals" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEBNext ARTIC SARS CoV2 RT PCR Module Manual</span></a><span>. </span></div></div>

[STEPS]
?. [cDNA Synthesis]
Gently mix and spin down the LunaScript RT SuperMix reagent. Prepare the cDNA synthesis reaction as described below:COMPONENTVOLUMERNA Sample8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µlFor no template controls, mix the following components: COMPONENTVOLUME(white) Nuclease-free Water8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µl
COMPONENTVOLUMERNA Sample8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µl
COMPONENTVOLUME(white) Nuclease-free Water8 µl
(lilac) LunaScript RT SuperMix2 µlTotal Volume10 µl
?. [cDNA Synthesis]
Incubate reactions in a thermocycler* with the following steps:CYCLE STEPTEMPTIMECYCLESPrimer Annealing25°C2 minutes1cDNA Synthesis55°C20 minutesHeat Inactivation95°C1 minuteHold4°C∞
CYCLE STEPTEMPTIMECYCLESPrimer Annealing25°C2 minutes1cDNA Synthesis55°C20 minutesHeat Inactivation95°C1 minuteHold4°C∞
Samples can be stored at   for up to a week.
?. [cDNA Amplification]
Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below:For Pool Set A: COMPONENTVOLUMEcDNA (Previous Step) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 1  1.75 µlTotal Volume12.5 µlFor Pool Set B: COMPONENTVOLUMEcDNA (Step 2) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 21.75 µlTotal Volume12.5 µl
4.5 µl of cDNA is recommended. If using less than 4.5 µl of cDNA, add nuclease-free water to a final volume of 4.5 µl. We recommend setting up the cDNA synthesis and cDNA amplification reactions in different rooms to minimize cross-contamination of future reactions. NEBNext VarSkip Short Primer Mixes cannot be combined with NEBNext ARTIC Primer Mixes in the same targeted amplification reaction.
COMPONENTVOLUMEcDNA (Previous Step) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 1  1.75 µlTotal Volume12.5 µl
COMPONENTVOLUMEcDNA (Step 2) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 21.75 µlTotal Volume12.5 µl
?. [cDNA Amplification]
Incubate reactions in a thermocycler* with the following steps:CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds1Denature95°C15 seconds35Annealing/Extension63°C5 minutesHold4°C∞1
CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds1Denature95°C15 seconds35Annealing/Extension63°C5 minutesHold4°C∞1
?. [Cleanup of cDNA Amplicons]
Vortex NEBNext Sample Purification Beads to resuspend.
The volume of NEBNext Sample Purification Beads provided here are for use with the sample composition at this step (25 μl; Step 5). These bead volumes may not work properly for a cleanup at a different step in the workflow, or if this is a second cleanup at this step. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.
?. [Cleanup of cDNA Amplicons]
Add  to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
[(0.8 X) resuspended beads]
?. [Cleanup of cDNA Amplicons]
Incubate samples on bench top for at least  at .
0 Room temperature
?. [Cleanup of cDNA Amplicons]
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant.
?. [Cleanup of cDNA Amplicons]
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Caution: do not discard the beads.
?. [Cleanup of cDNA Amplicons]
Add  to the tube/plate while in the magnetic stand. Incubate at  for , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
[80% freshly prepared ethanol]
0 Room temperature
?. [Cleanup of cDNA Amplicons]
Repeat the previous step once for a total of two washes: Add  to the tube/plate while in the magnetic stand. Incubate at  for , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
[80% freshly prepared ethanol]
0 Room temperature
?. [Cleanup of cDNA Amplicons]
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
?. [Cleanup of cDNA Amplicons]
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding . Elution volume can be adjusted for specific applications.
[0.1X TE]
?. [Cleanup of cDNA Amplicons]
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least  at . If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
0 Room temperature
?. [Cleanup of cDNA Amplicons]
Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer  to a new PCR tube. If elution volume was adjusted, the transfer volume should also be adjusted.
14 µl
?. [Cleanup of cDNA Amplicons]
We recommend assessing the cDNA amplicon concentrations with a size distribution with Qubit® fluorometer. Samples can be stored at –20°C for up to a week.
Amplicons may also be run on a Bioanalyzer or TapeStation® to confirm ~550 bp size of amplicons. To run on a TapeStation, dilute amplicon 10-fold with 0.1X TE Buffer and run 2 μl on a DNA High Sensitivity ScreenTape®. (See Figure 17 below for example of amplicon size profile on a Bioanalyzer).
?. [Cleanup of PCR Reaction]
Vortex NEBNext Sample Purification Beads to resuspend.
The NEBNext Sample Purification Beads added here is specific for samples suspended in the  buffer described at step 24. These volumes may not work properly for a cleanup at a different step in the workflow. For cleanups of samples contained in different buffer conditions, the volumes may need to be experimentally determined.
?. [Cleanup of PCR Reaction]
Follow Step Case A (Individual PCR Reactions) if you will normalize individual final libraries prior to pooling for sequencing. Option A is recommended for samples with a large range of SARS-CoV-2 viral genome copies input into the cDNA synthesis reaction (Step 1), such as a set of samples with inputs outside of the 100–100,000 genome copies range.Follow Step Case B (Pooled PCR reactions) if you are not normalizing individual final libraries prior to pooling for sequencing. Option B is recommended for samples with a small range of SARS-CoV-2 viral genome copies input into the cDNA synthesis reaction (Step 1), such as a set of samples with inputs within the 100–100,000 genome copies range. Otherwise, a sequencing depth of at least 1 million reads per library may be required.
?. [Cleanup of PCR Reaction]
Add  to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
[(0.7X) resuspended beads]
?. [Cleanup of PCR Reaction]
Incubate samples on bench top for at least  at .
0 Room temperature
?. [Cleanup of PCR Reaction]
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
?. [Cleanup of PCR Reaction]
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Caution: do not discard the beads.
?. [Cleanup of PCR Reaction]
Add  to the tube/plate while in the magnetic stand. Incubate at  for , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
[80% freshly prepared ethanol]
0 Room temperature
?. [Cleanup of PCR Reaction]
Repeat the previous step once for a total of two washes: Add  to the tube/plate while in the magnetic stand. Incubate at  for , and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
[80% freshly prepared ethanol]
0 Room temperature
?. [Cleanup of PCR Reaction]
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
?. [Cleanup of PCR Reaction]
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding .
[0.1X TE]
?. [Cleanup of PCR Reaction]
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least  at . If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
0 Room temperature
?. [Cleanup of PCR Reaction]
Place the tube/plate on the magnetic stand. After  minutes (or when the solution is clear), transfer  to a new PCR tube and store at .
15 µl
-20 °C
?. [Cleanup of PCR Reaction]
Assess the library size distribution with Agilent Bioanalyzer or TapeStation high sensitivity DNA reagents. The sample may need to be diluted before loading. A peak size of 200–400 bp is expected, based on a 25-minute fragmentation time (Figure 37).
If excess adaptor dimer peak is observed at 150-180 bp, a second 0.7X bead cleanup can be performed. The second 0.7X cleanup will result in slightly larger libraries which will not affect sequencing results.
?. [Cleanup of PCR Reaction]
Add 35 μl (0.7X) resuspended beads to the 50 μl pool. Mix well by pipetting up and down at least 10 times. Take care to expel all of the liquid out of the tip during the last mix. Vortexing for 3–5 seconds on high can also be used. If centrifuging pool after mixing, be sure to stop the centrifugation before the beads start to settle out.
For cleanup of pooled PCR reactions, we recommend cleanup of a 50 μl pool volume. For non-normalized pools using a 24-reaction kit, add 3 μl of each library to the pool, then mix, spin down, and take 50 μl of the pool to cleanup. For non-normalized pools using a 96-reaction kit, add 2 μl of each library to the pool, then mix, spin down, and take 50 μl of the pool on to cleanup. The amount of NEBNext Sample Purification Beads added here is specific for pooled samples suspended in the buffer described in this step. Using the exact amount of beads as described here may not work properly for sample cleanup at other steps in the workflow. If your samples are suspended in a different buffer at this point, the amount of beads needed may need to be experimentally determined.
?. [Cleanup of PCR Reaction]
samples on bench top for at least  minutes at room temperature.
?. [Cleanup of PCR Reaction]
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate well before placing on the magnetic stand.
?. [Cleanup of PCR Reaction]
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Caution: do not discard the beads.
?. [Cleanup of PCR Reaction]
Add 200 μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for  seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
?. [Cleanup of PCR Reaction]
Repeat the previous Step once for a total of two washes:Add 200 μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for  seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
?. [Cleanup of PCR Reaction]
Air dry the beads for up to  minutes while the tube/plate is on the magnetic stand with the lid open.Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the pool when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
?. [Cleanup of PCR Reaction]
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 25 μl of 0.1X TE.
?. [Cleanup of PCR Reaction]
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least  minutes at room temperature. If necessary, quickly spin the pool to collect the liquid from the sides of the tube or plate well before placing back on the magnetic stand.
?. [Cleanup of PCR Reaction]
Place the tube/plate on the magnetic stand. After   minutes (or when the solution is clear), transfer 23 μl to a new PCR tube and store at –20°C.
?. [Cleanup of PCR Reaction]
Assess the library size distribution with Agilent Bioanalyzer or TapeStation high sensitivity DNA reagents. The pool may need to be diluted before loading. A peak size of 200–400 bp is expected, based on a 25-minute fragmentation time (Figure 37).
If excess adaptor dimer peak is observed at 150–180 bp, a second 0.7X bead cleanup can be performed. The second 0.7X cleanup will result in slightly larger libraries which will not affect sequencing results.
?. [cDNA Amplification]
Combine the Pool A and Pool B PCR reactions for each sample.
Samples can be stored at –20°C for up to a week.Please consider the downstream application before proceeding. The next section lists instructions for a standard bead cleanup, however, it may not be necessary for your application.
?. [Cleanup of cDNA Amplicons]