TITLE: Modified Nucleic Acid Extraction Protocol for Humic-Rich Permafrost Peat Samples
AUTHORS: Yueh-Fen Li, Rhiannon Mondav, Isogenie 1 Project Coordinators, Virginia Rich

[DESCRIPTION]
Slight modifications to the manufacturer’s protocol (Qiagen #12966-10, now discontinued and replaced with #12988-10) enabled the successful extraction of high-quality DNA and RNA from permafrost peat samples. Integrating the DNA and RNA purification steps immediately after the extraction process helped streamline the workflow, resulting in consistent quality nucleic acids suitable for downstream sequencing and analysis. This improved approach optimizes the extraction process, ensuring more reliable and efficient results for permafrost peat studies.

[BEFORE_START]
To improve the chances of maintaining RNA integrity, perform the extraction using RNase-free practices. Be meticulous in cleaning your workspace, pipettes, and reagents with RNase-decontamination solutions such as RNaseZap. Ensure that all tubes and reagents are RNase-free and handle samples in a sterile environment using proper personal protective equipment (PPE). Additionally, minimize sample handling time at room temperature and work quickly to reduce the risk of RNA degradation.

[GUIDELINES]
To improve the chances of maintaining RNA integrity, perform the extraction using RNase-free practices. Be meticulous in cleaning your workspace, pipettes, and reagents with RNase-decontamination solutions such as RNaseZap. Ensure that all tubes and reagents are RNase-free and handle samples in a sterile environment using proper personal protective equipment (PPE). Additionally, minimize sample handling time at room temperature and work quickly to reduce the risk of RNA degradation.

[STEPS]
SECTION: Total nucleic acids extraction
1. Preheat the water bath to 60 ˚C. If the phenol:chloroform:isoamyl alcohol (PCI) (25:24:1, pH8.0) is refrigerated, move from 4 ˚C to the fume food to warm to room temperature (RT).  CAUTION: PCI is toxic and needs to be handled in the fume hood with proper PPEs. Handle with care.

SECTION: Total nucleic acids extraction
3. Add 8 to 10 g of peat slurry (i.e., the peat preserved in Lifeguard) to the 50-ml bead tube (which is part of the kit). Note: in the PowerMax manufacturer’s protocol, if Lifeguard is used on a sample, it is recommended to spin down the samples and remove it before adding the bead tube. However, we observed residual nucleic acids in the removed Lifeguard, and found good extraction efficiency when keeping Lifeguard in the slurry, so we keep it in; it is possible that the diluting effect of porewater in these often-saturated samples mitigates any negative impact on extractions of keeping Lifeguard in.
 

SECTION: Total nucleic acids extraction
4. In the fume hood, add 15 ml PM1 to the sample-containing bead tube, then add 5 ml PCI. Shake to mix. (Note, with PCI, be sure to take reagent from the lower organic layer).
SECTION: Total nucleic acids extraction
5. Ensure cap firmly tightened, and place the tube securely onto the vortex adapter of the Vortex-Genie 2 mixer inside the fume hood. Vortex at maximum speed for 10 min.
SECTION: Total nucleic acids extraction
6. Centrifuge at 4,500 x g for 6 min at RT. Transfer the aqueous phase of supernatant to the new 50 ml tube. Record the volume (about 18-24 ml).
SECTION: Total nucleic acids extraction
7. Add 1/3 volume (of the transferred supernatant) of C3 (about 6-8 ml), invert four to six times to mix. Place tubes on ice for 5 min.
SECTION: Total nucleic acids extraction
8. Centrifuge at 4,500 x g for 6 min at RT. Transfer supernatant into two 50 ml tubes (about 10-12.5 ml each).
SECTION: Total nucleic acids extraction
9. Add 1 volume of the transferred volume of PM3 (about 10-12.5 ml) and 1 volume of absolute ethanol (10-12.5 ml). Invert four to six times to mix.
SECTION: Total nucleic acids extraction
10. Transfer the sample to the column tube. Centrifuge at 4,500 x g for 2 min at RT. Discard the flow-through and repeat till all liquid has been processed.
SECTION: Total nucleic acids extraction
11. Add 16 ml of C5 to the column, and centrifuge at 4,500 x g for 2 min at RT. Discard the flow-through.
SECTION: Total nucleic acids extraction
12. Add 16 ml absolute ethanol to the column, and centrifuge at 4,500 x g for 2 min at RT. Discard the flow-through. Repeat this wash step one more time.
SECTION: Total nucleic acids extraction
13. Dry the column by an additional centrifuge at 4,500 x g for 8 min at RT.
SECTION: Total nucleic acids extraction
14. Place the column in a new 50 ml tube. Leave the cap off for 10 min in the fume hood to allow remaining ethanol to evaporate.
SECTION: Total nucleic acids extraction
15. Add 5 ml nuclease-free water to the column, sit the tube at RT for 1 min, then centrifuge at 4,500 x g for 2 min at RT. After the nucleic acids are eluted, discard the column.
SECTION: Ethanol precipitation of total nucleic acids
16. Add 1/10 volume of 3M sodium acetate (pH 5.2) (5 ml), swirl to mix.
SECTION: Ethanol precipitation of total nucleic acids
17. Add 2.5-3 volume of cold absolute ethanol (kept at -80 ˚C freezer) (bring up to about 20 ml). Invert four to six times to mix.
SECTION: Ethanol precipitation of total nucleic acids
18. Leave the tubes at -80 ˚C overnight for total nucleic acids precipitation.
 

SECTION: Recover and concentrate the total nucleic acids
19. Pre cool the centrifuge to 4 ˚C, take out the samples from -80 ˚C to let it thaw a little bit, and take out PCI to let it get back to RT in the fume hood (kept in dark).
SECTION: Recover and concentrate the total nucleic acids
20. Centrifuge the sample at 4,500 x g at 4 ˚C for 35 min. Carefully decant the supernatant.
SECTION: Recover and concentrate the total nucleic acids
21. Wash the pellet with 5 ml cold 70 % ethanol (kept at -20 ˚C freezer). Centrifuge at 4,500 x g at 4 ˚C for 15 min. Decant the supernatant.
SECTION: Recover and concentrate the total nucleic acids
22. Invert tubes on kimwipe on a cleaned surface in the fume hood to air dry the pellet (takes about 15 min).
SECTION: Recover and concentrate the total nucleic acids
23. Resuspend the total nucleic acids pellet in 105 µl of TE buffer. Pipet up the TE buffer to wash the tube wall (especially the button tapered part) and flick the tube to mix.
SECTION: Recover and concentrate the total nucleic acids
24. Aliquot to two 2 ml tubes: 35 µl for DNA recovery and 65 µl for RNA recovery. What’s left is for running the agarose gel to check the integrity of the total nucleic acids (optional).
SECTION: Recover and concentrate the total nucleic acids
22.1. Meanwhile, preheat the heat block to 37 ˚C, and take out reagents  from -20 ˚C and 4 ˚C and put on ice for the following DNA and RNA recovery steps. Make a 1 % agarose gel for nucleic acids quality check (optional). 
SECTION: DNA and RNA recovery
25. RNA recovery- DNase treatment.
SECTION: DNA and RNA recovery
25.1. Make a master mix for the DNase treatment.
  
   ItemVolume (µl)Master Mix for 6.5 reactions  (µl)10x DNase buffer1065RNaseOut213Nucleic acids65-DNase16.5ddH2O22143Total100227.5

SECTION: DNA and RNA recovery
25.3.  Incubate at 37 ˚C (heat block) for 25 min.
SECTION: DNA and RNA recovery
25.2. Aliquot 35 µl of the master mix to RNA recovery tube. Pipette up and down to mix.
26.  DNA recovery- RNase treatment
26.1. Add 1 µl of RNase A (10 mg/ml) to each DNA recovery tube. Flick to mix.
26.2.  Incubate at 37 ˚C for 20 min.
SECTION: PCI cleanup
27. Add 414 µl and 350 µl of TE buffer to the DNA recovery tube and the RNA recovery tubes respectively to make the sample volume to 550 µl each.
SECTION: PCI cleanup
28. Add 1 volume of PCI (550 µl) to the samples. Shake vigorously for 15 s to mix then let sit at RT for 2 min.
SECTION: PCI cleanup
29. Centrifuge at 18,000 x g for 3 min at RT. Carefully transfer the top aqueous layer to a new 2-ml tube (about 500 µl).
SECTION: PCI cleanup
30. Add 1 volume of chloroform to the samples. Shake vigorously for 15 s to mix then let sit at RT for 2 min.
 

SECTION: PCI cleanup
31. Centrifuge at 18,000 x g for 3 min at RT. Carefully transfer the top aqueous layer to a new 2-ml tube (about 450 µl).
SECTION: Ethanol precipitation of total nucleic acids
32. Add 1/10 volume of 3M sodium acetate (pH 5.2) (about 45 µl).
SECTION: Ethanol precipitation of total nucleic acids
33. Add linear acrylamide (5 mg/ml) to a final concentration of 10-20 µg/ml (about 1.5 µl). Flick to mix.
SECTION: Ethanol precipitation of total nucleic acids
34. Add 2.5-3 volume of cold 100 % ethanol (kept at -80 ˚C freezer) (about 1.3 ml). Invert to mix.
SECTION: Ethanol precipitation of total nucleic acids
35. Incubate at -80 ˚C for 30 min.
 

SECTION: Recover and concentrate DNA and RNA
36. Pre cool the microcentrifuge to 4 ˚C, and take out the samples from -80 ˚C.
SECTION: Recover and concentrate DNA and RNA
37. Centrifuge the sample at 18,000 x g at 4 ˚C for 20 min. Decant the supernatant.
SECTION: Recover and concentrate DNA and RNA
38. Wash the pellet with 1 ml cold 70 % ethanol (kept at -20 ˚C freezer). Centrifuge at 18,000 x g at 4 ˚C for 15 min. Decant the supernatant.
SECTION: Recover and concentrate DNA and RNA
39. Invert tubes on a kimwipe on a cleaned surface in the fume hood to air dry the pellet (takes about 10 min).
SECTION: Recover and concentrate DNA and RNA
40. Resuspend the DNA pellet and RNA pellet in 50 µl and 25 µl with TE buffer respectively. Wash the wall and flick to mix.
 

SECTION: Recover and concentrate DNA and RNA
41. Store the DNA and RNA at -80 ˚C.
SECTION: Total nucleic acids extraction
2. Warm solution PM1 in the pre-heated water bath for 15 min. Meanwhile, clean the workspace and pipettes with RNaseZap to remove RNAse contamination.