TITLE: NEBNext® Varskip Short ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®) E7658 Express Protocol with One  Clean-up Step 
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®), NEB #E7658S/L 24/96 reactions. </div><br/><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. This protocol does not include a cleanup step for each sample after cDNA synthesis and after adaptor ligation. Performing RNA input normalization prior to cDNA synthesis and targeted amplification and/or normalizing final libraries prior to sequencing promotes more even distribution of reads across libraries. Skipping RNA input normalization, final library normalization, and cleanups reduces hands on time but may require deeper sequencing depth to reach sufficient coverage of each sample.</div><br/><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></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]
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.Use of the NEBNext ARTIC Human Primer Mix 1 and 2 are optional. If used, the appropriate NEBNext ARTIC Human Primer Mix and NEBNext VarSkip Short SARS-Cov-2 Primer Mix should be combined prior to use. More specifically, NEBNext ARTIC Human Primer Mix 1 should be combined with NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1 and NEBNext ARTIC Human Primer Mix 2 with NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2. Mixing directions are listed below.
?. [cDNA Amplification]
Gently mix and spin down reagents. Prepare the split pool cDNA amplification reactions as described below:For Pool Set A: If using the NEBNext ARTIC Human Primer Mix and a 24 reaction kit, combine  with  in a new tube, vortex and spin down reagents. If using a 96 reaction kit, combine  with  in a new tube, vortex and spin down reagents. Use  for each Pool Set A reaction.COMPONENTVOLUMEcDNA (Step 2) 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: If using the NEBNext ARTIC Human Primer Mix and a 24 reaction kit, combine  with  in a new tube, vortex and spin down reagents. If using 96 reaction kit, combine  with  in a new tube, vortex and spin down reagents. Use  for each Pool Set B reaction.COMPONENTVOLUMEcDNA (Step 2) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 2 * 1.75 µlTotal Volume12.5 µl
[NEBNext ARTIC Human Primer Mix 1]
[NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1]
[ NEBNext ARTIC Human Primer Mix 1]
[NEBNext VarSkip Short SARS-CoV-2 Primer Mix 1]
[combined mix]
COMPONENTVOLUMEcDNA (Step 2) 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
[NEBNext ARTIC Human Primer Mix 2]
[NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2]
[NEBNext ARTIC Human Primer Mix 2]
[NEBNext VarSkip Short SARS-CoV-2 Primer Mix 2]
[combined mix]
COMPONENTVOLUMEcDNA (Step 2) 4.5 µl(lilac) Q5 Hot Start High-Fidelity 2X Master Mix6.25 µlNEBNext VarSkip Short SARS-CoV-2 Primer Mix 2 * 1.75 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
?. [cDNA Amplification]
Combine the paired Pool A and Pool B PCR reactions for each sample.
Samples can be stored at   for up to a week.
When cDNA amplification cleanup steps are skipped, the cDNA amplicon pool must be diluted before to proceeding to library preparation. Description of sample dilution is detailed below.
?. [Dilution of cDNA Amplicons]
Transfer 1.3 μl of the pooled cDNA amplicons to a fresh tube.
?. [Dilution of cDNA Amplicons]
Add 11.7 μl of 0.1X TE for a final volume of 13 μl.
?. [Dilution of cDNA Amplicons]
The pooled cDNA amplicons may be run on a TapeStation® to confirm 560 bp size of amplicons without cleaning up.Cleanup of cDNA amplicon pool is not needed for assessing on a TapeStation. To run on a TapeStation, dilute an aliquot of the pooled amplicons 10-fold with 0.1X TE Buffer and run 2 μl on a DNA High Sensitivity ScreenTape. (See Figure 9 below for example of amplicon size profile on a Tapestation)
?. [Fragmentation/End Prep]
FRAGMENTATION SIZE RANGEINCUBATION @ 37°COPTIMIZATION80 bp–300 bp25 min20-30 min
FRAGMENTATION SIZE RANGEINCUBATION @ 37°COPTIMIZATION80 bp–300 bp25 min20-30 min
Fragmentation occurs during a 37°C incubation step. A fragmentation time of 25 minutes should be sufficient for most samples to generate mainly ~120 bp fragments, however, the time may need to be optimized.
?. [Fragmentation/End Prep]
Ensure that the Ultra II FS Reaction Buffer is completely thawed. If a precipitate is seen in the buffer, pipette up and down several times to break it up, and quickly vortex to mix. Place  until use.There are short videos on NEB.com about handling the FS reagents: Preparing FS Reagents: https://www.neb.com/tools-and-resources/video-library/quick-tips---preparing-the-nebnext-ultra-ii-fs-dna-reaction-buffer-and-enzyme-mixMaking FS master mix: https://www.neb.com/tools-and-resources/video-library/quick-tips---preparing-nebnext-ultra-ii-fs-dna-reaction-buffer-and-enzyme-mix-master-mix
on ice
?. [Fragmentation/End Prep]
Vortex the Ultra II FS Enzyme Mix 5-8 seconds prior to use and place .
on ice
It is important to vortex the enzyme mix prior to use for optimal performance
?. [Fragmentation/End Prep]
Add the following components to a 0.2 ml thin wall PCR tube :COMPONENTVOLUME PER ONE LIBRARYARTIC SARS-CoV-2 cDNA (Step 8)13 µl(yellow) NEBNext Ultra II FS Reaction Buffer3.5 µl(yellow) NEBNext Ultra II FS Enzyme Mix1 µlTotal Volume17.5 µl
on ice
COMPONENTVOLUME PER ONE LIBRARYARTIC SARS-CoV-2 cDNA (Step 8)13 µl(yellow) NEBNext Ultra II FS Reaction Buffer3.5 µl(yellow) NEBNext Ultra II FS Enzyme Mix1 µlTotal Volume17.5 µl
?. [Fragmentation/End Prep]
Vortex the reaction for  and briefly spin in a microcentrifuge.
?. [Fragmentation/End Prep]
In a thermocycler* run the following program:TEMPTIME37°C25 minutes65°C30 minutes4°C∞
TEMPTIME37°C25 minutes65°C30 minutes4°C∞
If necessary, samples can be stored at  ; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
?. [Adaptor Ligation]
Add the following components directly to the FS Reaction Mixture:COMPONENTVOLUMEFS Reaction Mixture (Previous Step)17.5 µl(red) NEBNext Adaptor for Illumina** 1.25 µl(red) NEBNext Ultra II Ligation Master Mix*15 µlTotal Volume33.75 µl
COMPONENTVOLUMEFS Reaction Mixture (Previous Step)17.5 µl(red) NEBNext Adaptor for Illumina** 1.25 µl(red) NEBNext Ultra II Ligation Master Mix*15 µlTotal Volume33.75 µl
Do not premix adaptor with the Ligation Master Mix
?. [Adaptor Ligation]
Set a 100 µl or 200 µl pipette to 25 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance.
?. [Adaptor Ligation]
Incubate at  for  in a thermocycler with the heated lid off.
20 °C
?. [Adaptor Ligation]
Add  to the ligation mixture from the previous step.
[(red or blue) USER® Enzyme]
Steps 19 and 20 are only required for use with NEBNext Adaptors. USER enzyme can be found in the NEBNext Multiplex Oligos (www.neb.com/oligos).
?. [Adaptor Ligation]
Mix well and incubate at  for  with the heated lid set to ≥ 47°C.
37 °C
Samples can be stored   at  .Note: Only a portion of the ligation reaction (7.5 μl) will move forward to PCR enrichment.
?. [PCR Enrichment of Adaptor-ligated DNA]
Follow step-case A. if you are using the following oligos (10 μM primer):Use option A for any NEBNext Oligo kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes at 10 μM.Follow step-case B. if you are using the following oligos (10 μM primers):Use Option B for any NEBNext Oligo kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined. Primers are supplied at 10 μM.
?. [PCR Enrichment of Adaptor-ligated DNA]
Add the following components to a sterile strip tube:COMPONENTVOLUMEAdaptor Ligated DNA Fragments (Step 20)7.5 µl(blue) NEBNext Library PCR Master Mix 12.5 µlIndex Primer/i7 Primer*,**2.5 µlUniversal PCR Primer/i5 Primer*,**2.5 µlTotal Volume25 µl
COMPONENTVOLUMEAdaptor Ligated DNA Fragments (Step 20)7.5 µl(blue) NEBNext Library PCR Master Mix 12.5 µlIndex Primer/i7 Primer*,**2.5 µlUniversal PCR Primer/i5 Primer*,**2.5 µlTotal Volume25 µl
?. [PCR Enrichment of Adaptor-ligated DNA]
Set a 100 µl pipette to 20 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
?. [PCR Enrichment of Adaptor-ligated DNA]
Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds 1Denaturation98°C10 seconds 5*Annealing/Extension65°C75 seconds Final Extension65°C5 minutes1Hold4°C∞
CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds 1Denaturation98°C10 seconds 5*Annealing/Extension65°C75 seconds Final Extension65°C5 minutes1Hold4°C∞
?. [PCR Enrichment of Adaptor-ligated DNA]
Proceed to Cleanup of PCR Amplification in the next step.
?. [Cleanup of PCR Reaction]
The NEBNext Sample Purification Beads provided here are for use with the sample contained in the exact buffer at this step. 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]
Vortex NEBNext Sample Purification Beads to resuspend.
?. [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.
[(0.7 X) 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.
?. [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 5 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 38)
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.
?. [PCR Enrichment of Adaptor-ligated DNA]
Add the following components to a sterile strip tube:COMPONENTVOLUMEAdaptor Ligated DNA Fragments (Step 20)7.5 µl(blue) NEBNext Library PCR Master Mix 12.5 µlIndex Primer Mix*5 µlTotal Volume25 µl
COMPONENTVOLUMEAdaptor Ligated DNA Fragments (Step 20)7.5 µl(blue) NEBNext Library PCR Master Mix 12.5 µlIndex Primer Mix*5 µlTotal Volume25 µl
?. [PCR Enrichment of Adaptor-ligated DNA]
Set a 100 µl pipette to 20 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
?. [PCR Enrichment of Adaptor-ligated DNA]
Place the tube on a thermocycler and perform PCR amplification using the following PCR cycling conditions:CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds 1Denaturation98°C10 seconds 6*Annealing/Extension65°C75 seconds Final Extension65°C5 minutes1Hold4°C∞
CYCLE STEPTEMPTIMECYCLESInitial Denaturation98°C30 seconds 1Denaturation98°C10 seconds 6*Annealing/Extension65°C75 seconds Final Extension65°C5 minutes1Hold4°C∞
?. [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]
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]
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 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]
Pool PCR Reactions 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 in the next Step is specific for pooled samples suspended in the buffer described in Step 24. Using the exact amount of beads as described in the next step 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]
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.
?. [Cleanup of PCR Reaction]
Incubate 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 30 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 30 seconds, 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.
?. [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 39).
?. [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.