TITLE: POLAR Express: Pathogen-Oriented Low-cost Assembly & Re-sequencing
AUTHORS: Brian Glenn St Hilaire

[DESCRIPTION]
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[STEPS]
?. [RNA Extraction]
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [RNA Extraction]
Add  MagBead DNA/RNA Wash 1 and mix by using a plate shaker at  for .
100 µl
Centrifuge: 1300 33, 25 10
?. [RNA Extraction]
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [RNA Extraction]
Add  MagBead DNA/RNA Wash 2 and mix by using a plate shaker at  for .
100 µl
Centrifuge: 1300 33, 25 10
?. [RNA Extraction]
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [RNA Extraction]
Add  95-100% ethanol and mix by using a plate shaker at  for .
150 µl
Centrifuge: 1300 33, 25 10
?. [RNA Extraction]
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [RNA Extraction]
To elute DNA/RNA from the beads, add  DNase/RNase-Free Water and mix using a plate shaker at  for
9 µl
Centrifuge: 1300 33, 25 10
?. [RNA Extraction]
Pellet the beads and transfer  of supernatant into a new tube. The eluted DNA/RNA can be used immediately or stored frozen at .
7 µl
-80 °C
?. [RT-PCR]
Combine the following components into a thin-walled PCR tube.  Luna Universal Probe One-Step Reaction Mix (2X)  Luna WarmStart RT Enzyme Mix (20X) hCoV-2019/nCoV-2019 (V3) Primer Set mix (Primer pool 1 & 2) (100µm) Random Hexamers (100 µM) Viral RNA/DNA extract
10 µl
1 µl
1 µl
1 µl
7 µl
?. [RT-PCR]
Set up and run the following RT-PCR program.1. Reverse transcription:   at 2. Initial PCR activation:  at 3. 2-step PCR cycling (25X):    Denaturation:   at     Annealing & Extension:  at 4. Hold:
55 °C
95 °C
95 °C
63 °C
4 °C
?. [RT-PCR]
Add 0.7x volume of sparQ PureMag beads to each sample well in RT-PCR plate and mix gently by either flicking or pipetting. For example, add  sparQ PureMag beads to a  reaction. Note that the volume of oil is not taken into consideration given the oil is effectively inert. Then pulse centrifuge to collect all liquid at the bottom of the tube.
14 µl
20 µl
?. [RT-PCR]
Incubate for   at .
4 Room temperature
?. [RT-PCR]
Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [RT-PCR]
Keeping on the magnetic plate, add  of   freshly made  ethanol to the side of the wall opposite to the pellet and let sit for .
150 µl
4 Room temperature
?. [RT-PCR]
Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for   then remove any remaining ethanol.
?. [RT-PCR]
and repeat once.
?. [RT-PCR]
Add 11 µl of  Tris-HCl (Ph 8.0) and pipette to mix well. Incubate for  at .
37 °C
?. [RT-PCR]
Separate beads on the Agencourt SPRIPlate Super Magnet Plate for  or until the beads have pelleted.
?. [RT-PCR]
Pellet the beads and transfer 10 µl of supernatant containing SARS-CoV-2 amplicons into a new tube. The eluted DNA can be used immediately or stored frozen at .
-20 °C
?. [Hackflex Library Preparation]
Combine the following components into a thin-walled PCR tube.  5X Hacklfex Buffer (20mM Tris, 20 mM MgCl, 50% DMF)  Nuclease-Free Water Enrichment Bead-Linked Transposomes (eBLT) SARS-CoV-2 amplicons
4 µl
5.5 µl
0.5 µl
10 µl
?. [Hackflex Library Preparation]
Set up and run the following thermocycler program.1. Tagmentation:   at 2.  Hold:
55 °C
10 °C
?. [Hackflex Library Preparation]
Add  of Hackflex Stop Buffer (0.2% SDS) to the sample.
5 µl
?. [Hackflex Library Preparation]
Set up and run the following thermocycler program.1. Tagmentation:   at 2.  Hold:
25 °C
10 °C
?. [Hackflex Library Preparation]
Place the plates on the Agencourt SPRIPlate Super Magnet Plate and incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [Hackflex Library Preparation]
Keeping on the magnetic plate, add  of  Hackflex Wash Buffer (10% PEG 8000, 0.25 M NaCl, 10mM Tris-HCL pH 8.0, 0.1mM EDTA) gently mix and then let sit for .
30 µl
4 Room temperature
?. [Hackflex Library Preparation]
Separate beads on the Agencourt SPRIPlate Super Magnet Plate for  or until the beads have pelleted.
?. [Hackflex Library Preparation]
Avoid disturbing the bead pellet, carefully remove and discard HWB (10% PEG 8000, 0.25 M NaCl, 10mM Tris-HCL pH 8.0, 0.1mM EDTA).
?. [Hackflex Library Preparation]
and repeat once.
?. [Hackflex Library Preparation]
Resuspend  beads in the following  Indexing-PCR Master Mix NEBNext Q5U Master Mix (2X) IDT for Illumina DNA/RNA UD Indexes Nuclease-free Water
20 µl
10 µl
1 µl
9 µl
?. [Hackflex Library Preparation]
Set up and run the following Indexing-PCR program.1. Initial Denaturation:  at 2. 3-step PCR cycling (6X):    Denaturation:   at     Annealing:  at     Extension:  at 3. Final Extension:   at 4. Hold:
98 °C
98 °C
62 °C
65 °C
65 °C
4 °C
?. [Hackflex Library Preparation]
Add an equal volume (1:1) of sparQ PureMag beads to the library pool and mix gently by either flicking or pipetting. For example, add  sparQ PureMag beads to a   reaction. Then pulse centrifuge to collect all liquid at the bottom of the tube.
25 µl
25 µl
?. [Hackflex Library Preparation]
Incubate for   at .
4 Room temperature
?. [Hackflex Library Preparation]
Place the pool on a magnet and incubate for  or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.
?. [Hackflex Library Preparation]
Keeping the pool on the magnet, add  of   freshly made  ethanol to the side of the wall opposite to the pellet and let sit for .
200 µl
4 Room temperature
?. [Hackflex Library Preparation]
Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for   then remove any remaining ethanol.
?. [Hackflex Library Preparation]
and repeat once.
?. [Hackflex Library Preparation]
Add 25 µl of  TE Buffer (10mM Tris-HCL pH 8.0, 0.1mM EDTA) and pipette to mix well. Incubate for  at .
37 °C
?. [RNA Extraction]
and repeat once.
?. [RT-PCR]
Add  of mineral oil to each RT-PCR reaction to avoid evaporation and subsequent reaction failure.
20 µl
?. [Hackflex Library Preparation]
After PCR, pool of each sample into a single tube and vortex to mix.
10 µl
?. [Quantifying Pool and Sequencing]
Quantify final pool with adapted libraries. Load onto sequencer using platform appropriate dilutions.
?. [RNA Extraction]
Without disturbing the pellet, transfer   of saliva sample and 1X DNA/RNA to the bottom of a well in a 96-well deep-plate of each sample to a new tube
25 µl
?. [RNA Extraction]
For each saliva sample recieved add equal volume saliva and 2X DNA/RNA Shield from the Quick-DNA/RNA Viral magbead kit and vortex. Centrifuge the samples at   to bring down debris.
Centrifuge: 500 33, 5 min
?. [RNA Extraction]
Add  1X DNA/RNA Shield supplemented with  of Proteinase K (20mg/mL) to each sample.Briefly mix by using a plate shaker at  for  and incubate   at .
25 µl
2.5 µl
Centrifuge: 1300 33, 25 10
0 Room temperature
?. [RNA Extraction]
Remove   of MagBinding Beads per sample of the stock and place on a magnet stand. Incubate for   or until the beads have pelleted and the supernatant is completly clea. Then while avoiding the bead pellet, carefully remove the clear supernatnat.  Resuspend the beads in    of Viral DNA/RNA Buffer  (2-Mercaptoethanol 0.5% (v/v)) per sample and vortex to fully resuspend
5 µl
100 µl
2-Mercaptoethanol is toxic, causing irritation to the nasal passageways and respiratory tract upon inhalation, irritation to the skin, vomiting and stomach pain through ingestion, and potentially death if severe exposure occurs.
?. [RNA Extraction]
Add  of the Viral DNA/RNA Buffer (2-Mercaptoethanol 0.5% (v/v)) and MagBinding Beads misture to each  sample in 1X DNA/RNA Shield. Mix by using a plate shaker at  for .
100 µl
50 µl
Centrifuge: 1300 33, 25 10
2-Mercaptoethanol is toxic, causing irritation to the nasal passageways and respiratory tract upon inhalation, irritation to the skin, vomiting and stomach pain through ingestion, and potentially death if severe exposure occurs.