TITLE: F-2 FECES PROCESSING
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol details feces processing.

[BEFORE_START]
BEFORE START

Pre-cool the Bullet Blender by adding dry ice into the cooling compartment and running the cooling program. 
Pre-heat the heater mixer at 95 °C. 
Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% /molecular biology grade ethanol to remove additional contaminants.
Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 1) to Thermo Scientific Screw Cap Micro 1.5 ml Tubes.
For the first time use of IndiMag pathogen kit, add 100% ethanol to Buffer AW1 and AW2, and add 100% isopropanol to ACB as indicated on the bottles.
MagAttract Suspension G from IndiMag pathogen kit needs to be vortexed thoroughly for 3 min (before first use) or 1 min (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended. 
Aliquot nuclease-free water in big bottle into a few 15 ml tubes for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.


[GUIDELINES]
OBJECTIVE

To outline procedures for total nucleic acid extraction from fecal samples.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on procedures for total nucleic acid extraction from fecal samples to provide materials for downstream library preparation and sequencing for pathogen detection. 

RESPONSIBLE PERSON

Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
 

MAINTENANCE OF EQUIPMENT

Caution on RNA handling

RNases are very stable and difficult to inactivate, and only minute amounts are sufficient to destroy RNA. 
Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure. 
Sample handling and extraction should be performed under an extraction hood and respecting Good Laboratory Practices. 
Use filter tips all the time.

Storage of the buffers from IndiMag Pathogen Kit

Proteinase K is stable for at least 1 year after delivery when stored at Room temperature (15-25°C). To store for more than 1 year or if ambient temperature often exceeds 25 °C, storage at 2-8 °C is recommended. Do not add Proteinase K directly to the Buffer VXL mixture! This can cause clogs or precipitates.
Precipitation may form after storage at low temperature or prolonged storage. To dissolve precipitate, incubate Buffer VXL or ACB for 30 min at 37 °C, with occasional shaking.
Reconstituted Buffer AW1 can be stored at Room temperature (15-25°C) for up to 1 year. Mix well after adding Ethanol.
Buffer AVE is RNase-free upon delivery. It contains sodium azide, an antimicrobial agent that prevents growth of RNase-producing organisms. However, as this buffer does not contain any RNase degrading chemicals, it will not actively inhibit RNases introduced by inappropriate handling. When handling Buffer AVE, take extreme care to avoid contamination with RNases. Follow general precautions for working with RNA, such as frequent change of gloves and keeping tubes closed whenever possible.


APPENDICES

 
APPENDIX 1. MEASURING SPOON FOR 0.1 MM BEATING BEADS

The spoon (Next Advance, MSP01-RNA) is used for 0.1 mm beating beads measurement. One scoop equals to 100 µL.
 APPENDIX 3. Expected Outcomes


      SampleAmountSample conditionElution volumeDNA conc. (ng/ul)RNA conc.(ng/ul)Fecal 100 mg Frozen/Fresh755-505 - 100

 
 

[STEPS]
SECTION: 1. SAMPLE LYSIS
1. Add 700 µL of ASL buffer to the bead tubes prepared in step 4 from Before Start under the Guidelines & Warning tab.

SECTION: 1. SAMPLE LYSIS
2. Use a clean spatula or forceps to weigh about 100 mg fecal sample (roughly 5 drops of mouse feces) and place it into each prepared bead tube (If fecal sample is frozen, thaw and keep it on ice). Record the weight.
SECTION: 1. SAMPLE LYSIS
3. Include a positive control for each batch of samples: transfer 37.5 µL ZymoBIOMICS Microbial Community Standard Material, 100 µL EBV, and 100 µL ZIKV standard into a tube from step 4 from Before Start. Add 250 µL ASL buffer.
SECTION: 1. SAMPLE LYSIS
4. Include a negative control for each batch of samples: a bead tube from step 4 from Before Start with 500 µL ASL buffer only.
SECTION: 1. SAMPLE LYSIS
5. Refill the dry ice compartment of the Bullet Blender if necessary. Load all samples into the Bullet Blender.
SECTION: 1. SAMPLE LYSIS
6. Set the speed at 12 and the time at 3. Press Start.
SECTION: 1. SAMPLE LYSIS
7. After beating, incubate samples at 95 °C for 15 min.

SECTION: 1. SAMPLE LYSIS
8. After incubation, beat the samples in the Bullet Blender (Refill the dry ice compartment if necessary) at speed 12 and time at 3.
SECTION: 1. SAMPLE LYSIS
9. Let the samples settle for 1 min in the Bullet Blender and then repeat step 8. 



SECTION: 2. INSTRUMENT SET UP
10. 
 
Confirm 96 deep-well magnetic heads and 96 well deep-well heat blocks are being used.
SECTION: 2. INSTRUMENT SET UP
11. Ensure the program IndiMag_Pathogen_KF_Flex_4wash or the program has been downloaded and loaded onto the KingFisher Flex instrument.
SECTION: 3. SET UP THE PROCESSING PLATES
12. Set up the Tip Comb, Wash, and Elution Plates outside the instrument according to the following table. 
 
  
     Plate   ID      Plate   position      Plate   type      Reagent      Volume   per well        Tip comb      7      Place a 96 Deep-well Tip comb in a deep-well   plate        Elution      6      Deep-Well      Nuclease-free   water      75   µL        Wash 4      5      Deep-Well      100   % ethanol      750   µL        Wash 3      4      Deep-Well      80%   ethanol      750   µL        Wash 2      3      Deep-Well      Buffer   AW2      700   µL        Wash 1      2      Deep-Well      Buffer   AW1      700   µL        Sample       1      Sample   Lysate      Lysate   and lysis buffer      985   µL   

SECTION: 4. EXTRACTION
13. Centrifuge the bead tubes with lysate from step 9 for 12000 x g, 5 min. 

SECTION: 4. EXTRACTION
14. Add 20 µL of Proteinase K into wells of a Deep-well plate (based on the number of samples).
SECTION: 4. EXTRACTION
15. Transfer 270 µL supernatant without any particle carryover to the wells of the Deep-well plate containing proteinase K. This plate becomes the Sample Plate.
SECTION: 4. EXTRACTION
16. Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix; avoid foaming (it can be mixed on a Hula mixer for 2 min). Add 695 µL mixture to each sample. Each well including sample lysate should be 985 µL.

SECTION: 4. EXTRACTION
17. Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
SECTION: 4. EXTRACTION
18. Start the run, then load the prepared plates into position when prompted by the instrument.
SECTION: 5. QUANTIFICATION AND STORAGE
19. After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage. 
SECTION: 5. QUANTIFICATION AND STORAGE
20. In a 0.6-mL microcentrifuge tube, use 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions.
 
 
SECTION: 5. QUANTIFICATION AND STORAGE
21. Proceed with sample testing following the REDI-NET SOP F-4 Fecal Testing or store at -20 °C for less than 2 weeks. 
 

SECTION: 6. INSTRUMENT SET UP
22. 
 
Confirm 12-tip magnetic heads and 12 well deep-well heat blocks are being used.
SECTION: 6. INSTRUMENT SET UP
23. Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
SECTION: 7. SET UP THE SAMPLE PLATE AND ELUTION STRIP
24. Set up the Sample Plate according to the table below:  
     Row   ID      Plate   Row      Reagent      Volume   per well        Sample row      A      Lysate   and lysis buffer      985   µL        Wash 1      B      Buffer   AW1      700   µL        Wash 2      C      Buffer   AW2      700   µL        Wash 3      D      80   % ethanol      750   µL        Wash 4      E      100   % ethanol      750   µL        Tip Comb       F      Tip   comb                       G      Empty               H   

SECTION: 7. SET UP THE SAMPLE PLATE AND ELUTION STRIP
25. Set up the Elution Strip according to the table below: 
 
 
     Strip   ID      Row         Reagent      Volume   per well        Elution         A      Nuclease-free   water      75   µL   

SECTION: 8. EXTRACTION
26. Centrifuge the bead tubes with lysate from step 9 for 12000 x g, 5 min.

SECTION: 8. EXTRACTION
27. Add 20 µL of Proteinase K into wells of Row A (based on number of samples) of a new Deep-well plate.

SECTION: 8. EXTRACTION
28. Transfer 270 µL supernatant without any particle carryover to the wells of the Deep-well Row A containing proteinase K. This row becomes the Sample Row.

SECTION: 8. EXTRACTION
29. Add 135 µL Buffer VXL, 540 µL Buffer ACB, and 20 µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming. Add 695 µL mixture to each sample. Each well including sample lysate should be 985 µL.

SECTION: 8. EXTRACTION
30. Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
SECTION: 8. EXTRACTION
31. Start the run, then load the prepared plate and Elution Strip into position when prompted by the instrument.

 

SECTION: 9. QUANTIFICATION AND STORAGE
32. After the running protocol is completed (~35 minutes), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.
SECTION: 9. QUANTIFICATION AND STORAGE
33. In a 0.6-mL microcentrifuge tube, use 1 µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions. 
 

SECTION: 9. QUANTIFICATION AND STORAGE
34. Proceed with sample testing following the REDI-NET SOP F-4 Fecal Testing or store at -20 °C for less than 2 weeks. 
 

SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
35. DNA quantification: 
According to the volume of sample used, add the 1x HS dsDNA Qubit Assay for a final volume of 200 µL (i.e., if using 1 µL of sample, add 199 µL of 1x HS dsDNA Qubit Assay). Vortex for 5 - 10 seconds, then incubate for 2 min at Room temperature before reading. 
SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
36. RNA Quantification:
SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
36.1. In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay:   
     Reagents      Volume/sample      Volume   for n+1 sample        Qubit RNA HS Assay buffer      199   µL      ….   µL        Qubit RNA HS Assay Dye      1   µL      ….   µL   
 
SECTION: APPENDIX 2. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
36.2. In a new 0.6 ml tube, mix 199 µL of Qubit HS RNA Assay working solution and 1 µL of the sample. Vortex for 5 - 10 seconds, then incubate for 2 min at Room temperature before reading.