TITLE: Nucleic acid extraction - RNeasy PowerMicrobiome Kit (Qiagen)
AUTHORS: Adélaïde Roguet, Shuchen Feng

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Total nucleic extraction from wastewater using RNeasy PowerMicrobiome kit (qiagen)</div></div>

[STEPS]
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For HA filter extraction, add 6.5 µL of beta-Mercaptoethanol to each of the 2-mL ZR BashingBead Lysis tubes. Go to step 2. For BCoV/BRSV extraction, add 5 µL of BCoV/BRSV solution to the 2-mL tube containing the warmed PM1 + beta-Mercaptoethanol solution. Vortex for 15 seconds (speed 7 out of 10) and let sit for 10 min. Skip step 2.For Direct extraction, add 150 µL of wastewater to the 2-mL tube containing the warmed PM1 + beta-Mercaptoethanol solution. Vortex for 15 seconds (speed 7 out of 10) and let sit for 10 min. Skip step 2.
Add the beta-Mercaptoethanol while samples are still frozen. Let thaw at room temperature.
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For HA filter extraction, place the 2-mL tubes in the bead beater.
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Bead beat for
Start the bead beating when the beads start to be loose in the tubes.
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Cooldown the samples on ice for  .
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Repeat Steps 9.1 and 9.2 once .
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Centrifuge at maximum speed for 1 min at room temperature.
Centrifuge: 150000 33, 1 min
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For HA filter extraction, transfer 450 µL of supernatant to a Collection Tube (provided in the RNeasy PowerMicrobiome kit). For BCoV/BRSV/Direct extraction, transfer all supernatant.
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For HA filter/BCoV/BRSV extraction, add 150 μL of Solution IRS.For Direct extraction, add 100 μL of Solution IRS.
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Centrifuge at maximum speed for 1 min  . Avoiding the pellet and transfer the supernatant to a new Collection Tube.
Centrifuge: 150000 33, 1 min
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Add 650 μL each of Solution PM3 and Solution PM4. Vortex by inverting the tubes 15 times.
Visually, check that the 3 solutions are well mixed.
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Load the mixture into an MB Spin Column.
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Shake well to mix Solution PM5 and add 650 μL to the MB Spin Column.
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Centrifuge at max speed for 1 min .
Centrifuge: 150000 33, 1 min
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Discard flow-through. Add 600 μL of Solution PM4.
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Centrifuge at max speed for 1 min .
Centrifuge: 150000 33, 1 min
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Discard flow-through and centrifuge filter at max speed for an additional 2 min .
Centrifuge: 150000 33, 2 min
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Place the MB Spin Column in a clean 2-ml Collection Tube (provided in the RNeasy PowerMicrobiome kit).
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Add 60 μL of RNase-Free Water (warmed to 55ºC) to the center of the MB Spin Column membrane. Incubate at room temperature for 5 min  .
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Centrifuge at max speed for 1 min . Discard the MB Spin Column.
Centrifuge: 150000 33, 1 min
The DNA/RNA is now ready for downstream applications. RNA extract may be stored in RNase-free water at -80ºC for 1 year.
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Vortex briefly to mix (speed 7 out of 10). Incubate at 2–8°C for 5 min.
Place the tubes in a cold rack (stays in the refrigerator).
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Using centrifuge: - Load 650 μL of the mixture into an MB Spin Column- Centrifuge at max speed for 1 min . Discard the flow-through and repeat until all the mixture has been loaded onto the MB Spin Column.
Centrifuge: 150000 33, 1 min
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Using manifold: - Load the all mixture into an MB Spin Column (see picture)- Centrifuge at max speed for 1 min . Discard the flow-through.
Centrifuge: 150000 33, 1 min