TITLE: Extraction of RNA from Wastewater Primary Solids Using a Direct Extraction Method for Downstream SARS-CoV-2 RNA Quantification
AUTHORS: Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, Alexandria Boehm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><h1>Overview</h1></div><div class = "text-block">This SOP describes pre-analytical procedures to be followed for the isolation and identification of SARS-CoV-2 RNA in primary settled solids samples from wastewater treatment plants. This protocal should be paired with our digital PCR protocal. </div><div class = "text-block">The protocol follows the approximate workflow:</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Separate and aliquot sample (~4 hrs)</li><li style = "counter-reset:ol0;">Spike with  BCoV (~1 hr)</li><li style = "counter-reset:ol0;">Extract RNA (~ 12 hours total, may be split into 2-3 steps/days)</li><li style = "counter-reset:ol0;">Inhibitor removal (~1 hr)</li></ol></div><div class = "text-block"><h2>Biosafety Concerns</h2></div><div class = "text-block">Concentration and extraction procedures that utilize raw samples must adhere to strict Biosafety Level 2+ procedures. These procedures should be performed in a dedicated room. Downstream products may be handled using standard laboratory safety guidelines. </div><div class = "text-block">This protocol was developed by Stephanie Loeb, Katy Graham,  Marlene Wolfe,  Krista Wigginton,  and Alexandria Boehm at Stanford University and University of Michigan. </div></div>

[STEPS]
?. [Separate Solids and Spike BCoV]
Centrifuge at .
Centrifuge: 14500 33, 30 min, 4 10
Bring the centrifuge rotor to the BSC before loading or opening.
?. [Separate Solids and Spike BCoV]
Decant the supernatant into an empty 50 mL falcon tube or larger container. Record the weight and volume of the remaining solids.
Discard liquid as biohazard waste.
?. [Separate Solids and Spike BCoV]
Transfer solids for percent solids measurement: Transfer a generous (~) aliquot of the solid fraction onto a pre-weighed aluminum weigh dish. The aluminum weigh dish should be used directly out of the oven and not allowed to rehydrate. Record the total weight of the weigh dish before covering with aluminum and transferring to the oven to measure dry weight following EPA method Method 106.3. (Page 62) Link to EPA method. See page 62.
500 mg
If there is less than 2 g of solids available, % solids may be performed with a smaller aliquot.
?. [Separate Solids and Spike BCoV]
Aliquot solids as follows into tarred or pre-weighed tubes using a sterile spatula. Note exact mass aliquoted, with a target range from  to .
2 g
2.5 g
?. [Separate Solids and Spike BCoV]
Individually spike each sample that has been aliquoted in a 15mL PowerBead tube with 20 µL BCoV stock. Pipet the spike directly into the solids and mix well with a spatula to homogenize. Equilibrate the sample at 4°C for 1 hour.
Vials of BCoV vaccine should be rehydrated with molecular grade water before use via needle and syringe, and the solution may be stored at 4°C. For each batch of BCoV, a known dilution in PBS should be processed and analyzed in order to determine the concentration spiked
?. [Separate Solids and Spike BCoV]
Store samples in PowerBead tubes at  until extraction. Archive samples in cryovials at .
-80 °C
-80 °C
?. [Extract RNA]
Prepare for extractions using the RNeasy PowerSoil Total RNA kit to extract and isolate viral RNA into 100uL of eluent. This extract can be used for one step ddPCR or reverse transcribed for qPCR.Before starting  Prepare a sterile workspaceHeat Solution IRS at   until any precipitate dissolves.  Wear RNase-free gloves at all times and remove RNase from the work area. Heat Solution IRS at  until any precipitate dissolves.
60 °C
All sample processing must occur in the biosafety cabinet until the bead beating step is completed. After bead beating, cells have been completely lysed and the samples are considered non-infectious.
?. [Extract RNA]
Remove samples from the freezer and place . Samples do not need to be thawed before processing. Use Solution IRS while it is still warm.
on ice
?. [Extract RNA]
For processing samples already in PowerBead tubeAdd  of PowerBead Solution,of Solution SR1 and of Solution IRS directly to the tube containing the sample. Vortex the tube vigorously to suspend the solids.
2.5 mL
0.25 mL
0.8 mL
If the sample is not in a powerbead tube, perform step 11 in the sample tube, transferring the entire mixture to the PowerBead tube prior to step 12. If a large amount of residual solids remain in the tube after step 11, perform step 4 in two “washes” of 1.75 mL each phenol/chloroform/isoamyl alcohol. If a disposable spatula was used, the spatula can also be rinsed in this solution as well.
?. [Extract RNA]
Cap and vortex the PowerBead Tube to mix until the biphasic layer disappears. Place the PowerBead Tube on a Vortex Adapter and vortex at maximum speed for .
?. [Extract RNA]
Remove the PowerBead Tube and centrifuge at  for  at . After centrifugation, two or three phases may be visible. Total nucleic acids are retained in the upper aqueous phase.
Centrifuge: 2500 34
8 Room temperature
?. [Extract RNA]
Transfer the upper aqueous phase  to a clean 15 ml Collection Tube.  Take care not to transfer material from the lower phases.
After transferring the upper aqueous, the remaining fractions can be discarded as BSL2 waste.
?. [Extract RNA]
Add  of Solution SR3 to the aqueous phase and vortex to mix. Incubate at  for and then centrifuge at  for  at .
1.5 mL
4 °C
Centrifuge: 2.500 34
8 Room temperature
?. [Extract RNA]
Transfer the supernatant, without disturbing the pellet, to a new 15 ml Collection Tube.
?. [Extract RNA]
Add  of Solution SR4 to the supernatant in the Collection Tube and invert or vortex to mix. Incubate at  for .
5 mL
-20 °C
Samples can also be incubated at    , to continue procedure on the next day.
?. [Extract RNA]
Centrifuge at  for .
Centrifuge: 2500 34
?. [Extract RNA]
Decant the supernatant and invert the 15 ml Collection Tube on a paper towel for .
Check that the pellet seems to be firmly in the bottom of the tube - if so, you can carefully pour off the supernatant to decant.
?. [Extract RNA]
Shake Solution SR5 to mix and add  to the 15 ml Collection Tube. Resuspend the pellet completely by repeatedly pipetting or vortexing.
1 mL
The pellet will be difficult to resuspend. Place the tube in a heat block or water bath at   for  , followed by vortexing and/or pipetting up and down. Repeat until the pellet is resuspended. When the pellet is fully re-suspended, the solution will appear as a relatively opaque homogenous cloudy yellow-white solution. Continue heating and vortexing until no visible precipitates remain. Failing to fully re-suspend the pellet will result in large downstream losses of nucleic acids.
?. [Extract RNA]
Prepare one JetStar Mini Column from the RNeasy kit for each RNA isolation sample. Remove the cap of a 15 ml Collection Tube (provided) and place the JetStar Mini Column inside it. The column will hang in the Collection Tube.
?. [Extract RNA]
Add the RNA isolation sample from Step 21 onto the JetStar Mini Column and allow it to gravity flow through the column into the 15 ml Collection Tube.
Expect the flow-rate through the column to be slow (and highly variable between samples) - reserve enough time accordingly. If possible, allow samples to flow through under gravity only. If time to flow exceeds 1 hr, add positive pressure to the column using a syringe plunger. Do not force the flow at a rate of >1 drop per second.
?. [Extract RNA]
Add  of Solution SR5 to the JetStar Mini Column and allow it to completely gravity flow into the 15 ml Collection Tube.
1 mL
Apply positive pressure with syringe plunger to samples that are stalled. Do not force the flow at a rate of >1 drop per second.
?. [Extract RNA]
Transfer the JetStar Mini Column to a new 15 ml Collection Tube. Shake Solution SR6 to mix and then add to the JetStar Mini Column to elute the bound RNA. Allow Solution SR6 to gravity flow into the 15 ml Collection Tube.
1 mL
Apply positive pressure with syringe plunger to samples that are stalled. Do not force the flow at a rate of >1 drop per second.
?. [Extract RNA]
Transfer the eluted RNA to a 2.2 ml Collection Tube. Add of Solution SR4. Invert at least once to mix and incubate at  for a minimum of .
1 mL
20 °C
Samples can also be incubated at    , to split continue the procedure on the next day.
?. [Extract RNA]
Centrifuge the 2.2 ml Collection Tube at  for  to pellet the RNA.
Centrifuge: 13000 34
?. [Extract RNA]
Decant the supernatant and invert the 2.2 ml Collection Tube onto a paper towel for  to air dry the pellet.
?. [Extract RNA]
Resuspend the RNA pellet in  of Solution SR7. Observe the pellet carefully to make sure it is fully re-suspended. The RNA is now ready for downstream applications.
100 µl
Observe the base of the tube while slowly rotating the sample - there should not appear to be any material adhered to the side or base of the tube, and the solution should flow smoothly.
?. [Inhibitor Removal]
Use the Zymo OneStep PCR Inhibitor Removal Columns to remove inhibitors that were co-concentrated with the nucleic acids from the extracts to prevent downstream issues.
?. [Inhibitor Removal]
Centrifuge collection tubes in columns at for .
Centrifuge: 8000 34
?. [Inhibitor Removal]
Discard collection tube with flow through and load column into a labeled 1.5mL LoBind tube.
?. [Inhibitor Removal]
Add the full volume of RNA/DNA extract () to each column.
100 µl
?. [Inhibitor Removal]
Centrifuge LoBind tubes with column at  for
Centrifuge: 16000 34
?. [Inhibitor Removal]
This extract can be used for cDNA synthesis or as template for a one-step PCR reaction. Immediately perfom Nanodrop analysis, Qubit analysis, and/or reverse transcriptase if desired, or store eluent at  for later use.
-80 °C
?. [Separate Solids and Spike BCoV]
One 2g aliquot can be place directly into a 15 mL PowerBead tubes from RNeasy PowerSoil Total RNA extraction kit to prepare for extraction. Depending on the number of replicates required, additional aliquots can also be placed in PowerBead tubes for extraction.
?. [Separate Solids and Spike BCoV]
Additional 2g aliquots may be placed in as many 5mL cryovials as possible for storage at  .
-80 °C
?. [Separate Solids and Spike BCoV]
Load  samples of primary sludge at a time into the centrifuge rotor. Balance with equally weighted tubes filled with water.
40 mL
?. [Before Starting]
Day before sample processing:Thaw sample  at . Record the weight and volume of the sample.Day of sample processing:Place aluminum dishes in oven  before useSet centrifuge temperature to  before use
4 °C
4 °C
?. [Extract RNA]
Add  of phenol/chlorogorm/isoamyl alcohol (pH 6.5–8.0, not included in kit) to the sample tube. Vortex vigorously.
3.5 mL
?. [Extract RNA]
Add  of Solution SR5 to the JetStar Mini Column. Allow it to completely gravity flow through the column and collect in the 15 ml Collection Tube.
2 mL
Do not allow the column to dry out before loading sample in the next step.
?. [Inhibitor Removal]
Place one column into one collection tube for each sample you need and add  of Prep solution to each column.
600 µl