TITLE: Integra Total Nucleic Acid Extraction
AUTHORS: Sophana Chea, Sreyngim Lay, Mengheng Oum, Gechlang Tang, Cheata Hou, Manu Vanaerschot, Christina Yek, Jessica Manning, Vida Ahyong

[DESCRIPTION]
This SOP is the process of extracting Total Nucleic Acid (TNA) from Sera and/or Nasopharyngeal or Nasal swabs. The isolated high-quality nucleic acids are suitable for Next-Generation Sequencing (NGS).

[GUIDELINES]
Adapted from the Quick-DNA/RNA Pathogen MagBead Kit Manual (Zymo Research, Cat# R2145)

[STEPS]
SECTION: Reagent preparation (required with new kit)
1. 1. Add 20 mL of isopropanol to the MagBead DNA/RNA Wash 1 concentrate.
2. Add 30 mL of isopropanol to the MagBead DNA/RNA Wash 2 concentrate.
3. Add 1.2 mL of Proteinase K Storage Buffer per vial to reconstitute the lyophilized Proteinase K at 20 mg/mL 
Vortex to dissolve. STORE AT -20 °C  Freezer.

SECTION: Preparation the buffer plate (before starting protocol)
2. 1. Pre-make pathogen buffer plate with 880 µL Pathogen DNA/RNA buffer in 1ml deep well plate.
2. Pre-make bead plate with 25 µL MagBinding beads into 96V-well PCR plate.
*Make immediately before starting, <1h prior to starting the protocol, to ensure the beads are mixed.
3. Pre-make DNA/RNA Wash 1 plate with 550 µL Wash 1 buffer into a 1ml deep well plate.
4. Pre-make DNA/RNA Wash 2 plate with 550 µL Wash 2 buffer into a 1ml deep well plate.
5. Pre-make 100% ethanol plate with 1 mL  100% ethanol into a 2ml deep well plate.
6. Pre-make 80% ethanol plate with 600 µL 80% ethanol into a 1ml deep well plate.
7. Pre-make water plate with 50 µL DNAase/RNAse-free water in a 96 V-well PCR plate.
8. Pre-make water plate with 30 µL DNAase/RNAse-free water in a 96 V-well PCR plate.

Spin all plates down for 1 min except the bead plate. Perform a quick pulse spin down of the bead plate, just enough to get all liquid down. Centrifuge the rest of the plates at 12 000 rpm for 1 min .

SECTION: Sample preparation and Proteinase K
3. 1. Create a plate map so you know which sample you are adding to each well. Add 400 µL of your samples to 2ml deep well plate (Plate 1).
2. Manually add 65 µL of Proteinase K to each well of 8 well PCR strip tubes. Using a manual multichannel pipet, aliquot 4 µL of Proteinase K into each sample (Plate 1).
3. Load a set of Integra tips (tip set 1) onto the Integra.
4. Program: Pipet/Mix 250ul, 15 cycles, speed 10. Program the Integra to pipet 250 µL of your samples up and down for 1 min (15 cycles), then incubate at 25 Room temperature for 15 min .
*Keep tips.

SECTION: Sample binding and washing
4. 5. Program: Pipet 300ul. add 800 µL total of Pathogen DNA/RNA Buffer to the sample plate (Plate 1).
6. Program: Pipet/Mix 250ul, 30 cycles, speed 10. Program the Integra to mix samples and buffer for 2 min .         Keep tips.
7. Program: Pipet/Mix 20ul, 20 cycles. Program the Integra to mix the MagBinding beads plate so the beads are fully resuspended. If beads are not fully to the bottom of theplate, perform a short pulse spin.
8. Program: Pipet 20ul. Program the Integra to aspirate 20 µL from the MagBinding beads plate. Check that all wells have beads!
9. Program: Pipet/Mix 250ul, 30 cycles x 5 (10 min total), speed 7. Program the Integra to mix the beads with the sample for 10 min total. Keep tips.
10. Place the 96-well magnetic stand underneath the sample plate for 5 min until a bead ring forms.
11. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into a 2ml deep well waste plate.
Discard tips, load new tips.
12. Remove magnetic stand.
13. Program: Pipet 250ul. Dispense a total of 500 µL Wash 1 into the sample plate. Keep tips.
14. Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with the Wash buffer for 2 min total. Keep tips.
15. Place the 96-well magnetic stand underneath the sample plate for 2 min until bead ring forms.
16. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
17. Program: Pipet 250ul. Dispense a total of 500 µL Wash 2 into the sample plate. Keep tips.
18. Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with the wash buffer for 2 min  total. Keep tips.
19. Place the 96-well magnetic stand underneath the sample plate for 2 min  until a bead ring forms.
20. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Discard tips, load new tips.
21. Remove magnetic stand.
22. Program: Pipet 250ul. Dispense a total of 500 µL 100% Ethanol into the sample plate.
23. Program: Pipet/Mix 250ul, 30 cycles, speed 7. Program the Integra to mix the beads with Ethanol for 2 min total. Keep tips.
24. Place the 96-well magnetic stand underneath the sample plate for 2 min until a bead ring forms.
25. Program: Manual Pipet 300ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate.
26. Remove magnetic stand.
27. Repeat steps 22 to 26 for another round of Ethanol wash. Discard tips after 2nd Ethanol wash. Load new tips.
28. Keep the sample plate on the magnetic stand after the final Ethanol wash until the beads are dry (~10 min ).
29. Program: Pipet/Mix 33ul, 30 cycles, speed 7. Pipet 33 µL of Nuclease-free water (Heat the water at 55 °C for 10 min before elution) into the dried beads and mix the beads for 2 min total. Keep tips.
30. Place the 96-well magnetic stand underneath the sample plate for 2 min until a bead ring forms.
31. Program: Manual Pipet 30ul. Pipet 30 µL from the sample plate and dispense into a new 96 V-bottom PCR plate.
32. Store TNA sample immediately at -80 °C .

SECTION: DNase treatment post-TNA purification
5. 1. Aliquot SPRI beads (well mixed and resuspended) 50 µL into a new 96 V-bottom PCR plate.
2. Prepare DNase I solution
    a. Add 275 µL  DNase/RNase-free water to reconstitute lyophilized DNase I and mix.
3. Aliquot 20 µL  of TNA into a new 96 V-bottom PCR plate.
4. Prepare DNase master mix:
    a. 1x rxn = 20ul sample + 2.5ul DNase I + 2.5ul Digestion Buffer.
    b. Using a manual multichannel pipet, aliquot 5 µL of DNase I master mix to each sample and mix.
    c. Program: Pipet/Mix 20ul, 20 cycles, speed 7.
5. Incubate 15 min Room temperature 

SECTION: SPRI bead clean-up
6. 1. Program: Pipet/Mix 45ul, 20 cycles, speed 7. Add 45 µL SPRI beads (1.8x ratio) to the DNase I treated sample and mix by pipetting. 
2. Incubate 5 min at Room temperature .
3. Place the 96-well magnetic stand underneath the sample plate for 2 min until a bead ring forms.
4. Program: Manual Pipet 100ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
5. Program: Pipet 200ul. (1st Ethanol Wash). Aspirate and dispense 200 µL of freshly made 80% Ethanol into sample plate.
6. Incubate for 1 min at Room temperature .
7. Program: Manual Pipet 200ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Keep tips.
8. Program: Pipet 200ul. (2nd Ethanol Wash). Aspirate and dispense 200 µL of freshly made 80% Ethanol into sample plate.
9. Incubate for 1 min at Room temperature .
10. Program: Manual Pipet 200ul. Aspirate and discard the cleared supernatant into the 2ml deep well waste plate. Discard tips, load new tips.
11. Incubate for 5 min at Room temperature until beads are dry.
12. Remove plate from magnetic stand.
13. Program: Pipet/Mix 22ul, 10 cycles, speed 7. Add22 µL DNase/RNase-free water to each well and mix beads.
14. Place the 96-well magnetic stand underneath the sample plate for 2 min until a bead ring forms.
15. Program: Manual Pipet 20ul. Aspirate 20 µL of purified TNA sample to a new 96 V-bottom PCR plate. 
16. Store sample at -80 °C immediately.