TITLE: SARS-CoV-2 RNA extraction with Ceres Nanotrap and Zymo Environ Water 
AUTHORS: Amanda Windsor, kathryn.judy, Tamara Walsky, Padmini  Ramachandran, Chris  Grim, Maria Hoffmann

[DESCRIPTION]
This protocol uses the Ceres Nanotrap® particle-based virus capture and concentration method for 10mL of wastewater followed by extraction with the Zymo Environ™ Water RNA extraction kit with a Zymo DNase step. 

[BEFORE_START]
Store the reagents separately from RNA/TNA (total nucleic acid) samples. 
Use a clean designated work area and separate pipettes for pre- and post-extraction steps to minimize the potential for cross-contamination 
Wear a lab coat and protective eyewear. 
Wear gloves and change them often.
Prevent contamination by using aerosol-resistant pipette tips. 

[GUIDELINES]
When developing this protocol, we extracted at least 2 replicates of each wastewater sample to ensure we had ample RNA for downstream processes

RNA extraction is performed at room temperature and centrifugation at 10,000-16,000 x g for 30s 

[STEPS]
SECTION: Viral Capture with Nanotrap® Particles
3. Shake wastewater bottle to mix then let sit 45 s 

SECTION: Viral Capture with Nanotrap® Particles
4. Using a 10mL serological pipette, carefully pipette 10 mL of wastewater into a 15mL conical tube

 

SECTION: Viral Capture with Nanotrap® Particles
5. Add 100 µL of Nanotrap® Enhancement Reagent 2 (ER2) and invert 15mL tube 2-3 times to mix

SECTION: Viral Capture with Nanotrap® Particles
6. Re-suspend Nanotrap® particles by inverting the bottle 5 times

SECTION: Viral Capture with Nanotrap® Particles
9. Place samples on magnetic rack to separate Magnetic Nanotrap® particles from the sample - at least 2 min 

SECTION: Viral Capture with Nanotrap® Particles
10. After beads have settled, use a 5mL serological pipette to remove all of the supernatant without disturbing the pelleted beads


SECTION: Viral Capture with Nanotrap® Particles
11. Add 1 mL  of DNAse/RNAse Free water to the tube

SECTION: Viral Capture with Nanotrap® Particles
11.1. Remove tube from magnet and re-suspend the pelleted beads using a 100-1000uL pipette 
SECTION: Viral Capture with Nanotrap® Particles
11.2. Transfer suspended beads to a 1.5mL microcentrifuge tube 
SECTION: Viral Capture with Nanotrap® Particles
12. Place microcentrifuge tube on the 2mL tube-compatible magnetic rack 
SECTION: Viral Capture with Nanotrap® Particles
12.1. Incubate until the beads have settled - at least 2 min 

SECTION: Viral Capture with Nanotrap® Particles
None. Add 150 µL  of Nanotrap® particles to the sample tube and invert 2-3 times to mix do not vortex
SECTION: RNA Extraction with Zymo Environ™ Water RNA Kit
12. Remove tubes from magnet and Re-suspend the pelleted beads in 375 µL of Zymo DNA/RNA Shield™ and 125 µL Zymo DNAse/RNAse-Free water from the Zymo Environ™ Water RNA Kit

SECTION: RNA Extraction with Zymo Environ™ Water RNA Kit
13. Incubate the samples at 95 °C for 5 min 

SECTION: RNA Extraction with Zymo Environ™ Water RNA Kit
13.1. While tubes are incubating, add 400 µL RNA Binding Buffer to new 1.5mL microcentrifuge tubes, one tube per sample.

SECTION: RNA Extraction with Zymo Environ™ Water RNA Kit
14. Place tubes  from step 12 on magnetic rack and allow beads to settle approximately 2 min or until the solution is clear

SECTION: RNA Extraction with Zymo Environ™ Water RNA Kit
15. Transfer 400 µL of supernatant to corresponding 1.5mL tubes prepared in step 12.1 

SECTION: Viral Capture with Nanotrap® Particles
13. Remove supernatant with a 100-1000uL pipette without disturbing the pellet. Remove any small amount of remaining supernatant with a smaller pipette tip (e.g. 2-20uL pipette) 


SECTION: Viral Capture with Nanotrap® Particles
14. Remove the tubes from the magnet and re-suspend the pellet with 375 µL Zymo DNA/RNA Shield and 125 µL Zymo DNase/RNase-Free water from the Zymo Environ Water RNA Kit 
SECTION: Viral Capture with Nanotrap® Particles
15. Incubate the samples at 95 °C for 5 min 

SECTION: Viral Capture with Nanotrap® Particles
15.1. While samples are incubating, add 400 µL Zymo RNA Binding Buffer from the Zymo Environ Water RNA Kit to new 1.5mL tubes (one per sample)

SECTION: Viral Capture with Nanotrap® Particles
16. Remove tubes from heat block and place on a magnetic rack and allow beads to settle until supernatant is clear - at least 2 min


 

SECTION: Viral Capture with Nanotrap® Particles
17. Transfer 400 µL of supernatant to the corresponding tube prepared in step 15.1 and mix by gentle pipetting 
 


SECTION: Viral Capture with Nanotrap® Particles
16.1. Reset heat block temperature to 27 °C 

SECTION: Zymo Environ™ Water RNA Kit
18. Transfer entire sample to a Zymo-Spin™ IIICG Column in a clean collection tube and centrifuge 10000 x g, 30 s and keep the flow-through

 

SECTION: Zymo Environ™ Water RNA Kit
19. Add 800uL of ethanol (95-100%) to the flow-through in the collection tube from step 18 and mix well by gentle pipetting
 

SECTION: Zymo Environ™ Water RNA Kit
20. Transfer 800 µL into a new Zymo-Spin™ IIICG Column in a clean collection tube and centrifuge 10000 x g, 30 s and discard the flow through

SECTION: Zymo Environ™ Water RNA Kit
20.1. Repeat step 19 with the remaining 800 µL of sample using the same collection tube

SECTION: Zymo Environ™ Water RNA Kit
22. Add 100 µL Zymo DNase/RNase-Free water directly to the column matrix and centrifuge 10000 x g, 30 s and keep the flow-through for step 24

SECTION: Zymo Environ™ Water RNA Kit
23. Place a Zymo-Spin III-HRC Filter into a new collection tube and add 600 µL Prep Solution Centrifuge 8000 x g, 3 min discard the flow-through

SECTION: Zymo Environ™ Water RNA Kit
23.1. transfer the column to an RNase-Free 1.5mL microcentrifuge tube
SECTION: Zymo Environ™ Water RNA Kit
24. Transfer the eluted RNA from step 22 into the Zymo-Spin III-HRC filter prepared in step 23 and 16000 x g, 3 min  keep the flow-through

SECTION: Zymo Environ™ Water RNA Kit
25. Add 200 µL RNA Binding Buffer to the filtrate and mix well by gently pipetting up and down.
SECTION: Zymo Environ™ Water RNA Kit
26. Transfer the mixture into a Zymo-Spin IC column in a collection tube and 10000 x g, 30 s discard the flow-through

SECTION: Zymo Environ™ Water RNA Kit
29. Add 400 µL of RNA Prep Buffer to the column and 10000 x g, 30 s discard the flow-through.
SECTION: Zymo Environ™ Water RNA Kit
30. Add 700 µL of RNA Wash Buffer to the column and 10000 x g, 30 s  discard the flow-through.

SECTION: Zymo Environ™ Water RNA Kit
31. Add 400 µL of RNA Wash Buffer to the column and 10000 x g, 2 min to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
SECTION: Zymo Environ™ Water RNA Kit
32. Add 50 µL of DNase/RNase-Free Water directly to the column matrix
and 10000 x g, 30 s The eluted RNA can be used immediately or stored at -70 °C.

SECTION: Zymo Environ™ Water RNA Kit
27. Add 400 µL RNA Wash Buffer to column and 10000 x g, 30 s Discard flow-through

SECTION: Zymo Environ™ Water RNA Kit
28. Add 5 µL DNase I and 35 µL DNA Digestion Buffer to the column matrix

SECTION: Zymo Environ™ Water RNA Kit
28.1. Incubate 27 °C   20 min 

SECTION: Before you start
1.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     Turn on heat block95 °C 
SECTION: Before you start
2. Ensure appropriate volume of DNase I is available (5µL per sample), or make new aliquots
SECTION: Before you start
2.1. Add 275 µL DNase/RNase-Free water to reconstitute lyophilized DNase I (1U/µL)

 

SECTION: Viral Capture with Nanotrap® Particles
7. Add 150 µL Nanotrap® particles to the sample

 

SECTION: Viral Capture with Nanotrap® Particles
8. Incubate samples Room temperature 10 min with constant rotation 

SECTION: Zymo Environ™ Water RNA Kit
21. Add 400 µL of RNA Prep Buffer to the column and centrifuge 10000 x g, 30 s and discard the flow-through
SECTION: Zymo Environ™ Water RNA Kit
21.1. Transfer column to an RNase-Free 1.5mL microcentrifuge tube
SECTION: Zymo Environ™ Water RNA Kit
25.1. Add 300 µL of ethanol (95-100%) to the filtrate + RNA Binding Buffer and mix well by gently pipetting up and down.