TITLE: V.4 - Direct wastewater RNA capture and purification via the "Sewage, Salt, Silica and SARS-CoV-2 (4S)" method
AUTHORS: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/ffce19a396882be605eb2c93185943f2745a7f7440126d7b6b804da64064cc05/cfqqbdz6x.png" /></div></div>

[STEPS]
?. [Preparing RNA wash buffers]
Prepareeach of two wash buffers - Wash buffer #1 (4S-WB1) and #2 (4S-WB2), for later use during cleanup of RNA bound to silica columns.
1 L
?. [Preparing RNA wash buffers]
4S-WB1 composition:   ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M1.5 M300 mL3Ethanol100%20%200 mL4TRIS pH 7.21 M10 mM10 mL5Pure water (MilliQ or distilled)NANA490 mLAddwater to sterile bottleAddofNaClAddof EthanolAddofTRISAgitate to fully mix buffer solution
ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M1.5 M300 mL3Ethanol100%20%200 mL4TRIS pH 7.21 M10 mM10 mL5Pure water (MilliQ or distilled)NANA490 mL
490 mL
300 mL
200 mL
10 mL
?. [Preparing RNA wash buffers]
4S-WB2 composition:   ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M100 mM20mL3Ethanol100%80%800mL4TRIS pH 7.21 M10 mM10mL5Pure water (MilliQ or distilled)NANA170mLAddwater to sterile bottleAddofNaClAddof EthanolAddofTRISAgitate to fully mix buffer solution
ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M100 mM20mL3Ethanol100%80%800mL4TRIS pH 7.21 M10 mM10mL5Pure water (MilliQ or distilled)NANA170mL
170 mL
20 mL
800 mL
10 mL
?. [Preparing RNA wash buffers]
Prepare two tubes containing lysis salts (one for the sample and one as a matched negative control) by addingof sodium chloride to each sterile 50mL tubes. MakeTE buffer (TRIS,EDTA). Addof TE buffer to each 50mL tube with salt. Gently shake.
9.35 g
400 µl
?. [Sample preparation, RNA preservation and particle lysis]
Obtain awastewater sample and pour directly into the pre-salted tube. Agitate sample until all NaCl dissolves in the wastewater. Maintain atduring transport to the lab.
40 mL
4 °C
Here, NaCl lyses lipid-protein envelopes, denatures proteins and disrupts RNA-protein interactions. EDTA inhibits the enzymatic degradation of RNA by RNases present in wastewater and TRIS provides optimal buffering conditions for nucleic acids.
?. [Sample preparation, RNA preservation and particle lysis]
Obtain sterile 1x PBS and pour directly into the second pre-salted tube. Agitate sample until all NaCl dissolves in the PBS. Maintain atduring transport to the lab. Perform same steps with the PBS-only negative control as described below for the wastewater sample.
40 mL
4 °C
?. [Sample preparation, RNA preservation and particle lysis]
Resuspend dry bovine coronavirus stock (Bovilis Coronavirus Calf Vaccine) inof PBS. Dilute this resuspended stock into PBS at a dilution of 1:10 (of stock into PBS). Spike 50uL of diluted  bCoV into the wastewater sample as a process control. Agitate sample to fully mix bCoV or other spiked-in controls with the wastewater sample.
2 mL
100 µl
900 µl
Other spike-in controls can be used instead of bCoV, such as Phi6 bacteriophage. In addition, purified RNAs can be used to quantify the extraction efficiency of "free RNA".
?. [Sample preparation, RNA preservation and particle lysis]
Heat of remaining bCoV spike-in aliquot atfor.  Freeze for later quantification via RT-qPCR. This enables more accurate assessment of the bCoV spike-in concentration.
200 µl
75 °C
?. [Sample preparation, RNA preservation and particle lysis]
(RECOMMENDED) Heat inactivate sample atfor. Our unpublished analyses have shown that this step may improve some RNA species' enrichment and detection.
70 °C
?. [Sample preparation, RNA preservation and particle lysis]
Filter the sample through a 5-um PVDF filter via syringe filtration or funnel top vacuum into a sterile 100mL tube.
?. [Direct RNA extraction (RNA Binding, Washing, Eluting)]
Add ofto theof filtrate.
40 mL
40 mL
?. [Direct RNA extraction (RNA Binding, Washing, Eluting)]
Agitate sample to mix ethanol and wastewater lysate.
?. [Direct RNA extraction (RNA Binding, Washing, Eluting)]
Attach Zymo III-P (or other) silica spin column to a vacuum manifold. Agitate the wastewater and lysate by inverting the tube five times, then pour into the spin column and vacuum the fullof wastewater lysate & ethanol through the spin column.
80 mL
Commercial silica spin columns vary in their silica membrane packing tightness, changing the flow rate of lysed wastewater. We advise the use of the Zymo III-P column to avoid column clogging issues, but columns such as the Qiagen RNeasy, QIAamp Mini Spin and Zymogen II-CR can act as substitutes, depending on vacuum strength and sample particulate content. Large-format "maxiprep" style columns are also able to purify wastewater RNA, but require a large volume RNA elution up to 20mL (Step 13) and a dowstream precipitation-concentration step (Isopropanol precipitation, see companion protocol, Step 12) .
?. [Direct RNA extraction (RNA Binding, Washing, Eluting)]
Vacuumwash buffer #1 (4S-WB1) through the silica spin column.
5 mL
?. [Direct RNA extraction (RNA Binding, Washing, Eluting)]
Vacuumwash buffer #2 (4S-WB2) through the silica spin column.
10 mL
?. [RNA elution]
Detach silica spin column from vacuum manifold, remove any attached reservoirs/funnels and place column into a 1.5-mL centrifugation-compatible flowthrough collection tube.
?. [RNA elution]
Centrifuge silica spin column in tube atto remove any residual 4S-WB2 present in the column.
Centrifuge: 10000 34, 2 min, 20 10
?. [RNA elution]
Discard the collection tube and place silica column into a new 1.5-mL centrifugation-compatible flowthrough collection tube.
?. [RNA elution]
Pre-warmof ZymoPURE elution buffer or TE buffer per RNA sample toin a heat block, waterbath or incubator.
200 µl
200 µl
50 °C
?. [RNA elution]
Addof pre-warmed elution buffer to each silica spin column. Incubate the elution buffer and column + collection tube assembly at room temperature for.
200 µl
?. [RNA elution]
Spin atto elute RNA from the column. The flowthrough present in the collection tube contains the purified RNA.
Centrifuge: 10000 34, 5 min, 20 10
?. [Storage]
The eluted RNA is now ready for downstream analysis. Store RNA atfor same-day use or freeze atfor later use and storage.
4 °C
-80 °C
?. [Waste disposal]
Liquid waste: Per sample, this protocol generates 90 mL of liquid waste containing 37.5% ethanol, 9.35 g NaCl, and trace amounts of Tris and EDTA. This should be treated as a hazardous chemical waste containing ethanol and should NOT be poured down the drain (see campus or local municipal guidelines).Biohazard waste: All consumables and solid waste (e.g. filters, gloves, tubes) used in this protocol should be treated as biohazardous waste.Any excess wastewater samples should be treated with bleach to final concentration of 10% v/v before removal from the biosafety cabinet for drain disposal.
?. [Troubleshooting   ]
In our hands, about 1% of Zymo III-P columns clog when passing the wastewater lysate through the columns. Wastewater that remains after the column clogs can slowly be pushed through the column using a luer lock syringe, but note that this impacts the speed at which wastewater passes through the column, and thus possibly the yield. Use caution when analyzing RNA from clogged columns.