TITLE: Wastewater grab sample processing with Nanotrap® Microbiome A particles (40 ml) 
AUTHORS: Dilip Abraham, Venkata Raghava Mohan, Nirmal Kumar

[DESCRIPTION]
This protocol is used to carry out processing of wastewater using Nanotrap‱ Microbiome A Particles (Ceres Nanosciences). Nanotrap‱particles are affinity-capture magnetic hydrogel particles that have been shown to capture and concentrate microorganisms (like SARS-CoV-2) from wastewater prior to RNA or TNA extraction. The sample is mixed with the particles, incubated, and separated with a magnet. The supernatant is removed, the pellet is spiked with extraction controls, and subjected to lysis followed by extraction. This protocol has been adapted from the online protocol available from Ceres Nanosciences.

[BEFORE_START]
Extraction control preparation
DNA control
Before first use of the sample process control (SPC), prepare a 1/10 dilution of the control DNA in nuclease free water and store them  at -70 °C as small aliquots to avoid freeze thaw. Add 1 µL of 1/10 diluted SPCC into particle pellet. 

RNA control
Prepare a 1/10 dilution of the MS2 extraction control in nuclease free water and store them  at -70 °C as small aliquots to avoid freeze thaw. Add 1 µL of 1/10 diluted MS2 into particle pellet. 

[GUIDELINES]
Processed sample pellet needs to be extracted on the same day so as to avoid any nucleic acid degradation. The Nanotrap‱ A particles and the pellet obtained after sample concentration should not be frozen.

[STEPS]
SECTION: Wastewater grab sample processing with Nanotrap® Microbiome A particles
1. Wastewater grab samples collected in 50 mL  centrifuge  tubes are transported to the laboratory in cold chain, maintaining a temperature of 4 °C .
2. Once received, place the sample tubes on a tube rack and leave at Room temperature for 10 minutes, allowing large aggregates to settle at the bottom of the tube.
3. Using a serological pipette, carefully transfer 40 mL of clear supernatant into a new 50 ml centrifuge tube without disturbing the sediment.
4. Add 400 µL of ER2 Enhancer reagent to the supernatant, invert 2-3 times and mix throughly. 
5. Add 600 µL  of Nanotrap‱ A particles to the above and invert mix 2-3 times. Incubate at Room temperature  for 20 min .
6. Place the tube on a  50 ml magnetic stand  for 10 minmin and allow the Nanotrap® particles to settle.
7. Carefully discard the supernatant without disturbing the pellet. If needed, use a pipette to remove all the supernatant from the tube.
8. Remove the tube containing pellet from the magnetic rack. The pellet is now ready for total nucleic acid extraction. The pellet should not be frozen, extraction is to be carried out on the same day.
9. Add 700 µL of the InhibitEX buffer along with 1 µL of 1/10 diluted SPC and 1 µL of 1/10 diluted RNA-MS2 control into each particle pellet and resuspend the pellet by vortexing the tube briefly.
10. Transfer the suspension into a 1.7 mLmicrocentrifuge tube. Incubate at Room temperature for 10 min .
11. Place the microcentrifuge tubes on a  1.5 ml magnetic separation rack until the Nanotrap® particles have settled, resulting in a clear solution.
12. Collect the supernatant into 2 mL microcentrifuge tube containing ~ 370 mgof acid-washed glass beads and discard the pellet.