TITLE: High-throughput SARS-CoV-2 RNA extraction from nasopharyngeal swabs using repurposed 3D printers v2.
AUTHORS: Koen  Vandelannoote

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here, we describe a semi-automated workflow for paramagnetic bead-based extraction of SARS-CoV-2 RNA from human upper respiratory samples that uses low-cost repurposed </span><span style = "font-style:italic;">Creality3D </span><span>Ender-3 3D-printers and readily accessible reagents. The protocol described here is for the use of four instruments in parallel, each processing 24 samples, 96 samples per batch.</span></div></div>

[STEPS]
?. [Pre-process sample information]
Scan the barcodes of 91 nasopharyngeal swabs using a barcode scanner and the "20200908_Ender_VX-500_extraction_template.xlsx" spreadsheet. Populate the “SampleID” column of the “Sample log” sheetRandomly include a negative extraction control for every set of 24 swabs in the sheet. Randomly include a PCR no-template-control (NTC) control in the sheet.
The spreadsheet will automatically parse 5 plate dispensing charts that can be printed for convenience:4 charts represent the plate layouts of the 4 Ender VX-500 extraction runs (of 24 samples each).1 chart represents the plate layout of the 96-well plate on which the eluates of the 4 extraction runs will be combined.
The "20200908_Ender_VX-500_extraction_template.xlsx" spreadsheet can be found as an attachment under the abstract tab.
?. [Pre-dispense the samples into a deep-well 96 well plate]
Transfer of swab transport medium into a deep-well 96 well plate in a Class II BSC using the plate layout charts as a guide.
100 µl
?. [Prepare four Ender VX-500 machines for extraction]
Attach three 8-tube PCR strips onto the “Tube holder”.
The caps of the strip fit tightly into the openings on the “Tube holder”.Only hold the strips by the caps and make sure not to touch the outside of the tubes.
?. [Prepare four Ender VX-500 machines for extraction]
Seat the “Tube holder” into the “Adaptor”.
?. [Prepare four Ender VX-500 machines for extraction]
Lock the “Tube holder” in place by inserting the provided Allan hex wrench. This will ensure the “Tube holder” is always inserted in the same position.
?. [Prepare four Ender VX-500 machines for extraction]
Place three “Magnetic bars” in the openings of the “Tube holder”.
?. [Prepare four 96-well plates for extraction]
Add  Lysis buffer to columns 1, 5 and 9 of a 96-well plates with V-shaped wells using a multichannel pipette.
18.5 µl
Resuspend any salted out guanidine thiocyanate crystals before dispensing the buffer.Incubate   for   .
?. [Prepare four 96-well plates for extraction]
Add  NFW to columns 4, 8 and 12 using a multichannel pipette.
80 µl
?. [Prepare four 96-well plates for extraction]
Add  to columns 2, 3, 6, 7, 10 and 11 using a multichannel pipette.
150 µl
[Ethanol]
The ethanol concentration will quickly lower in the air flow of the BSC. Always use a freshly made up Ethanol solution.
?. [Prepare four 96-well plates for extraction]
Transfer / pipet-mix of nasopharyngeal swab transport medium from the deep-well plate into columns 1, 5 and 9 using using a multichannel pipette and the relevant plate dispensing chart.
37 µl
?. [Prepare four 96-well plates for extraction]
Incubate the plate  for .
0 Room temperature
?. [Prepare four 96-well plates for extraction]
Transfer / pipet-mix SPRI bead mix into columns 1, 5 and 9 using a multichannel pipette.
30 µl
Shake the SPRI bead mix before use (the beads settle quickly).
?. [Prepare four 96-well plates for extraction]
Transfer / pipet-mix to columns 1, 5 and 9 using a multichannel pipette.
85.5 µl
[Ethanol]
?. [Prepare four 96-well plates for extraction]
Incubate the plate  for .
0 Room temperature
The prepared plates need to be extracted on the Ender VX-500 machines immediately after this incubation step.
?. [Extract 4 plates on 4 Ender VX-500 machines  ]
Insert the four 96-well plates onto four Ender VX-500 machines prepared for extraction
Ensure the microplates are correctly oriented by checking the position of the A1 marker.
?. [Extract 4 plates on 4 Ender VX-500 machines  ]
Power on the Ender VX-500 machines.
?. [Extract 4 plates on 4 Ender VX-500 machines  ]
Use the knob on the Ender VX-500 to start the extraction by selecting “Print from TF” and selecting “RNA_ex_v10.gcode”.
The "RNA_ex_v10.gcode" print job which emulates the RNA extraction can be found as an attachment under the abstract tab.
?. [Extract 4 plates on 4 Ender VX-500 machines  ]
The Ender VX-500 will:Collect bead-RNA complex from the lysate ()Wash 1: vortex () & collect bead-RNA complex ()Wash 2: vortex () & collect bead-RNA complex ()Air dry ()Elute: vortex () & collect beads ()
After  the machine will beep to indicate the run completed successfully.
?. [Extract 4 plates on 4 Ender VX-500 machines  ]
Remove the 96-well plates from the Ender VX-500 machines.
?. [Extract 4 plates on 4 Ender VX-500 machines  ]
Power off the Ender VX-500 machines.
?. [Extract 4 plates on 4 Ender VX-500 machines  ]
Place the 96-well plates on a 96 well magnetic rack and use a multichannel pipette to transfer of eluate onto a fresh 96-well plate that will combine the eluates of the 4 extraction runs.
50 µl
of eluate is sacrificed to prevent any bead SPRI bead carryover.
?. [Machine clean-up]
Carefully remove the "Tube holder" from the EnderVX-500 “Adaptor”.
?. [Machine clean-up]
Use paper tissues to release the three 8-tube PCR strips from the "Tube holder" and decontaminate the attachment in a bath containing a 1% sodium hypochlorite solution for min .