TITLE: SARS-CoV-2 Wastewater RNA Concentration and Extraction (Nanotrap® and NucleoMag® RNA Water)
AUTHORS: Brett Rasile, Kendra Maas

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Process </div></div>

[STEPS]
?. [Virus Capture & Concentration]
Collect  of wastewater in a 500mL or 1L plastic sample bottle.
500 mL
?. [Virus Capture & Concentration]
Create a 1:100 dilution of Zoetis Bovine Rhinotracheitis-Parainfluzena-Respiratory Syncytial Virus (BRSV) Vaccine in H2O.
?. [Virus Capture & Concentration]
Spike the wastewater sample with  of 1:100 BSRV to achieve a 1µL/mL concentration of 1:100 BSRV.
500 mL
500 µl
This can be applied to wastewater samples greater or less than 500mL, as long as a 1µL/mL concentration is achieved.
?. [Virus Capture & Concentration]
Mix the added 1:100 BRSV into the wastewater sample by aggressively inverting the bottle several times or shaking.
?. [Virus Capture & Concentration]
Incubate spiked wastewater samples for a minimum of  at room temperature (RT) to allow large aggregates to sediment at the bottom of the sample bottle.
?. [Virus Capture & Concentration]
Use a 25mL serological pipette to transfer top  of spiked wastewater to a 50mL conical tube.
40 mL
Make sure to gently transfer from the top of the wastewater sample as to not disturb the sedimented particles at the bottom of the bottle.
?. [Virus Capture & Concentration]
Addof Ceres Magnetic Nanotrap® particles to the   spiked wastewater aliquot.
600 µl
40 mL
?. [Virus Capture & Concentration]
Place conical tubes into custom magnet racks and allow magnets to attract Magnetic Nanotrap® particles for a minimum of 20 minutes at RT.
The samples should be allowed to sit on the magnets long enough so that the supernatant is relatively clear. Some wastewater samples may be too turbid to reach this point, however.
?. [Virus Capture & Concentration]
Keeping the conical tubes fixed to the magnets, pour off the supernatant carefully as to not disturb the pellet of Magnetic Nanotrap® particles at the bottom of the conical tubes.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Add  of Buffer MWA1 to the falcon tubes.
500 µl
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Vortex to resuspend the Magnetic Nanotrap® pellets in the Buffer MWA1.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Incubate samples for  at RT.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Place conical tubes on the custom magnet racks to separate the Magnetic Nanotrap® particles.
?. [Virus Capture & Concentration]
Invert the wastewater samples several times to incorporate the Magnetic Nanotrap® particles and incubate for 20 minutes at RT.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
With the tubes on the magnets, transfer  of lysate to a 2mL deep well plate.
450 µl
Use barcode labels on the falcon tubes to transfer sample ID's to a LIMS program when transferring lysate to the deep well plate.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Add  of Buffer MWA2 and   of NucleoMag® B-Beads to the lysate in the deep well plate.
475 µl
25 µl
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Shake the sample plate for  at  ,  .
33
56 °C
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Place the sample plate on a plate magnet for at least  to separate NucleoMag® B-beads.
At each magnet step, allow as much time as needed for the NucleoMag® B-beads to completely precipitate near the magnets.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Use a multichannel pipette to remove the supernatant from each well.
Ensure that NucleoMag® B-beads are not removed with the supernatant. If beads are drawn up with the supernatant, dispense the liquid back into the well and incubate on a magnet for a few minutes to allow the NucleoMag® B-beads to settle at the magnet.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Add of Buffer MWA3 to each well.
850 µl
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Shake the sample plate for  at  ,  .
33
56 °C
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Place the sample plate on a plate magnet for at least  to separate NucleoMag® B-beads.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Use a multichannel pipette to remove the supernatant from each well.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Add of Buffer MWA3 to each well.
850 µl
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Shake the sample plate for  at  ,  .
33
56 °C
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Place the sample plate on a plate magnet for at least  to separate NucleoMag® B-beads.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Use a multichannel pipette to remove the supernatant from each well.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Add of Buffer MWA4 to each well.
850 µl
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Shake the sample plate for  at  ,  .
33
56 °C
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Place the sample plate on a plate magnet for  to separate NucleoMag® B-beads.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Use a multichannel pipette to remove the supernatant from each well.
Take extra care to remove as much supernatant as possible at this step. Carryover of the wash buffer can cause PCR/qPCR/library prep to fail.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Place plate on shaker at  for approximately 30 minutes to allow beads to air dry.
56 °C
Ensure beads are thoroughly dry before proceeding, carry over EtOH from Buffer MWA4 can ruin downstream experiments.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Add  of RNase-free H2O to each well.
60 µl
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Shake the sample plate for  at  ,  .
33
56 °C
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Place the sample plate on a plate magnet for  to separate NucleoMag® B-beads.
?. [Viral RNA Extraction (Nucleomag® RNA Water)]
Transfer eluted RNA to a 96-well elution plate for further processing.