TITLE: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA
AUTHORS: Aaron Topol, marlene.wolfe , Krista Wigginton, Bradley White, Alexandria Boehm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This process instruction describes the steps for purification of nucleic acids from wastewater solids and preparation for downstream quantitative analysis with Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR). Due to the large quantities of substances that have inhibitory effects on PCR in wastewater samples, a subsequent PCR inhibitor removal step is required after nucleic acid purification. Both steps of the process are carried out in a 96-well plate format. </div><div class = "text-block"><span>This method uses the dewatered solids generated using this protocol: </span><a href="http://dx.doi.org/10.17504/protocols.io.btyqnpvw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses</span></a><span>. </span></div><div class = "text-block">RNA purification is carried out using a kit optimized for the purification of viral on for the Perkin Elmer Chemagic 360. Although only RNA is used in the downstream applications from this protocol, DNA is also eluted in this process. A crucial component of the purification kit are the magnetic particles coated with poly vinyl alcohol (M-PVA Magnetic Beads) which have a hydrophilic surface giving them an affinity for nucleic acids but not many other biological molecules. The workflow involves binding nucleic acids in a sample to the beads which are then transferred through a series of wash buffers to remove debris with a robotic head with magnetic rods.</div><div class = "text-block">The OneStep PCR Inhibitor Removal Kits are PCR inhibitor clean up kits that contain all the components needed for efficient removal of contaminants that can inhibit downstream enzymatic reactions (e.g. PCR and RT) from DNA and RNA preparations. The column matrices in these PCR inhibitor clean up kits have been specifically designed for the efficient removal of polyphenolic compounds, humic/fulvic acids, tannins, melanin, etc. from the most impure DNA and RNA preparations.</div><div class = "text-block">This process instruction applies to extraction of RNA from wastewater samples using the Chemagic™ Viral DNA/RNA 300 Kit H96 for the Perkin Elmer Chemagic 360 followed by PCR Inhibitor Removal with the Zymo OneStep-96 PCR Inhibitor Removal Kit.</div></div>

[STEPS]
?. [RNA Extraction with the Chemagic 360 Process Steps]
Using a wide orifice 1000 µL pipette tip, transfer  per well of homogenized sample (prepared as specified in High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses) to each well of the Sample Plate as follows:
300 µl
?. [Chemagic Clean Up]
Dispose of empty Tip Rack (Position 1), empty Magbead plate (position 2) and Deep Well Plate with tips in it (Position 6) in a red biohazard waste bin.
?. [Preparation]
If necessary, dissolve lyophilized Proteinase K in of nuclease free water following manufacturer directions.
11 mL
?. [Preparation]
Label one 50 mL conical tube as Lysis buffer.
?. [Preparation]
Label a deep-well-plate as “Sample Plate”.
?. [Preparation]
If the Chemagic 360 has not been used within 24 hours, run the protocol “Check Manifolds All.che” to prime the buffer manifolds.
.che files are protocols for the Chemagic 360 instrument and are available from Perkin Elmer.
?. [Preparation]
Elution Plates and Magbead Plates are generated in batches as needed with  Elution Buffer and  Magbeads per well. This can be done using a liquid handler such as the Agilent Bravo.
60 µl
150 µl
?. [Preparation]
If there is condensation on the seal of the prepared Elution Plate, briefly spin the plate to collect the buffer in the bottom of the well.
?. [Preparation]
With the plate sealed, resuspend the magnetic beads in the prepared Magbead Plate by shaking in the BioShake XP at  for
33
?. [Preparation]
Check that the Viral RNA Buffers are connected to the Chemagic tubing. If not, perform a normal cleaning procedure, switch to Viral RNA Buffers and prime the system.
?. [RNA Extraction with the Chemagic 360 Process Steps]
Using normal pipette tips:Add  of water to the indicated wells in the figure below to create extraction negative controls.Add  of gRNA SARS-CoV-2 control (at a concentration of 50 cp/µl) to the indicated wells to represent the positive extraction control.
300 µl
10 µl
?. [RNA Extraction with the Chemagic 360 Process Steps]
Mix the Lysis buffer according to the following volumes per sample/plate:ReagentsVolume per SampleVolume for Full Plate 
(with dead volume)Proteinase K10 µL1 mLLysis Buffer 1300 µL30 mL
ReagentsVolume per SampleVolume for Full Plate 
(with dead volume)Proteinase K10 µL1 mLLysis Buffer 1300 µL30 mL
?. [RNA Extraction with the Chemagic 360 Process Steps]
Add of Lysis buffer solution mixed in step 11 to each well of the Sample Plate.
310 µl
NOTE: The Proteinase K activity will decrease after incubation longer than   in Lysis Buffer. Ensure that all samples are mixed with Proteinase K / Lysis Buffer within the 10 min.
?. [RNA Extraction with the Chemagic 360 Process Steps]
Set up the plate deck in the Chemagic 360 as follows:Position 1: Rack with Disposable TipsPosition 2: Magbead plate, low-well-plate prefilled with 150 µL Magnetic BeadsPosition 3: Sample PlatePosition 4: Empty Deep Well Plate Position 5: Empty Deep Well PlatePosition 6: Empty Deep Well PlatePosition 7: Elution Plate, Deep Well Plate with 60 µL Elution BufferPosition 8: Empty
?. [RNA Extraction with the Chemagic 360 Process Steps]
Select the protocol: “chemagic Viral300 360 H96 drying prefilling VD141210.che”
This .che file is available from Perkin Elmer
?. [RNA Extraction with the Chemagic 360 Process Steps]
Make sure that all wells are selected and start the protocol.
?. [RNA Extraction with the Chemagic 360 Process Steps]
When the protocol is complete, immediately proceed to PCR Inhibitor Removal or seal the Elution Plate and store on ice.
?. [Chemagic Clean Up]
Wrap Deep Well Plates in Positions 3, 4 and 5 in a blue absorbent pad, seal with lab tape, and dispose of as chemical waste.
?. [RNA Extraction with the Chemagic 360 Process Steps]
Add  of DNA/RNA shield stock solution with BCoV spike-in from pre-analytical processing to one well of the Sample Plate to serve as an extraction positive control.
300 µl
?. [RNA Extraction with the Chemagic 360 Process Steps]
Using a wide orifice tip, add the samples one at a time to ensure that a full  is pulled up and no clumps prevent the aspiration of the sample.
300 µl
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Add RNA extracts to prepped Silicon-A™-HRC Plate
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Select “Zymo 01 - Prep Reagent to Spin Column.pro” in the Protocol to Run dropdown menu.
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Prepare Silicon-A™-HRC Plate
The Silicon-A™-HRC Plate and Elution Plate come in the Zymo OneStep PCR Inhibitor Removal Kit
All the processes used to apply the Zymo OneStep PCR Inhibitor Removal Kit can be automated using a liquid handling robot. We use the Agilent Bravo to implement this section of the protocol.
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Mount the Silicon-A™-HRC Plate onto an Elution Plate
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Add  Prep Solution to the wells on the Silicon-A™-HRC Plate by piercing the cover foil in the middle with the pipette tip.
150 µl
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Incubate for
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Centrifuge the plate at exactly  for .
Centrifuge: 3500 34
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Discard Elution Plate with prep solution.
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
While preparing the Silicon-A™-HRC Plate, allow the Chemagic Elution Plate (from step 16) to sit on the ALPAQUA Magnum EX Universal Magnet Plate for 2 minutes to pull down residual magnetic beads.
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Mount the prepped Silicon-A™-HRC Plate with the RNA extracts onto a new Elution Plate.
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Transfer the full volume of extracted RNA in the Chemagic Elution Plate to the Silicon-A™-HRC Plate.
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Centrifuge the plate at exactly  for .
Centrifuge: 3500 34
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Immediately seal the Elution Plate and store on ice until ddPCR analysis.
?. [PCR Inhibitor Removal with OneStep-96 PCR Inhibitor Removal Kit]
Discard the Silicon-A™-HRC Plate.