TITLE: Qiagen RNEasy PowerMicrobiome RNA extraction kit
AUTHORS: Michael Dan Siemon, Christelle Schang et al, Jessica Pardy, Richard Gibson, Dilan Joseph, Justin Donovan, christopher.degroot

[DESCRIPTION]
The samples were processed using the Qiagen RNeasy PowerMicrobiome kit with the modifications described by Schang et al., 2021. As a substitute for vortexing described in the kit protocol, bead-beating was used. 100ul of phenol-chloroform-isoamyl alcohol was added to the bead beating tubes before the addition of the membranes. Bead-beating was conducted 4x for 30s at 4 m/s. After pelletizing the samples with centrifugation, the supernatant was processed through spin columns. Spin columns were incubated with DNase Digestion Solution for 15 minutes to isolate only RNA. The RNA was eluted from the spin columns by passing 50 uL of DEPC water through twice. 

[STEPS]
SECTION: Qiagen RNEasy PowerMicrobiome RNA
4. As a substitute for vortexing described in the kit protocol, bead-beating was used. Bead-beating was conducted 4x for 30s at 4 m/s.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
2. Add 100 µl phenol–chloroform–isoamyl alcohol to a PowerBead Bead Tube, Glass 0.1 mm. Place 0.25 g stool or biosolid sample into the Bead Tube. 
SECTION: Qiagen RNEasy PowerMicrobiome RNA
1. Thoroughly mix wastewater sample then aliquot 40 mL into 50 mL Falcon tube. Centrifuge at 4500 x g for 20 min. Decant supernatant, assume 280 µl pellet.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
6. After pelletizing the samples with centrifugation (13,000 x g for 1 min), mix the supernatant with 650 µl each of Solution PM3 and Solution PM4. 
SECTION: Qiagen RNEasy PowerMicrobiome RNA
3. Add 650 µl PM1 and 6.5 µl β-mercaptoethanol to the PowerBead Tube
SECTION: Qiagen RNEasy PowerMicrobiome RNA
10. Incubate spin columns with DNase Digestion Solution for 15 minutes at room temperature to isolate only RNA.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
7. Process the solution through MB RNA Spin Column by centrifugation (13,000 x g for 1 min). Discard flow-through and repeat until all the solution has been processed. 
SECTION: Qiagen RNEasy PowerMicrobiome RNA
8. Shake solution PM5, add 650 µl to the Spin Column and centrifuge (13,000 x g for 1 min).
SECTION: Qiagen RNEasy PowerMicrobiome RNA
9. Conduct a drying step by centrifuging at 13,000 x g for 1 min to remove residual wash.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
15. Elute RNA from the spin columns into 1.5 ml eppendorf tubes by passing 50 uL of DEPC water through twice. Centrifuge each elution 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
12. Discard flow-through. Add 650 µl Solution PM5. Centrifuge at 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
13. Discard flow-through. Add 650 µl Solution PM4. Centrifuge at 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
11. Add 400 µl Solution PM7 and centrifuge at 13,000 x g for 1 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
14. Conduct a drying step by centrifuging at 13,000 x g for 2 min.
SECTION: Qiagen RNEasy PowerMicrobiome RNA
5. After pelletizing the samples with centrifugation (13,000 x g for 1 min), mix the supernatant with 150 µl Inhibitor Removal Solution in a new collection tube and vortex briefly to mix. Incubate at 2–8°C for 5 min.