TITLE: Low Volume Methodology for Nextera DNA Flex Library Prep Kit (96 Samples)
AUTHORS: Eric Adams, Stephen Wandro, Julio Avelar-Barragan, Andrew Oliver, Katrine Whiteson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol presents a low volume methodology for the Nextera DNA Flex Library Prep Kit (96 Samples). This method increases the number of sequencing libraries which can be generated using each kit from 384 samples (4 x 96-wells) to 864 samples (9 x 96-wells). While other low-cost methods have been deveolped for this purpose, this protocol is a very straightforward method utilizing reduced reagent and sample volumes.</div><div class = "text-block">Update: The "Nextera DNA Flex Library Prep" kit has been renamed to "Illumina DNA Prep" with the appropriate indexes having been similarly renamed. While all reagents and steps remain identical, an updated version of this protocol  may be published to account for any differences between the protocols.</div></div>

[STEPS]
?. [Preparation]
Equilibrate the following reagents at  for Bead-Linked Transposomes (BLT)Tagmentation Buffer (TB1)Tagment Stop Buffer (TSB)Tagment Wash Buffer (TWB)Sample Purification Beads (SPB)
0 Room temperature
?. [Preparation]
Thaw the Resuspension Buffer (RSB) and bring to  then vortex to mix
0 Room temperature
?. [Preparation]
Prepare a proteinase K solution at
For a 1mL solution: add 15μL of 20mg/mL proteinase K to 985μL of nuclease-free water
?. [Preparation]
Thaw KAPA HiFi HotStart ReadyMix (2X)
on ice
?. [Preparation]
Thaw the i5 and i7 indexes at
0 Room temperature
Index tubes: vortex and spin down brieflyIndex plates: spin down at   to ensure no droplets remain on the plate coverIndex stocks should be diluted to    working stocks
?. [Preparation]
Prepare a Tagmentation Master Mix (TMM)TMM contains 1μL BLT and 1μL TB1 per reactionVortex BLT vigorously before use and ensure the beads are evenly resuspended
?. [Preparation]
Prepare PCR Master Mix (PCR MM) PMM contains 2.75μL nuclease-free water, 0.5μL KAPA-PCR-F primer, 0.5μL KAPA-PCR-R primer, and 6.25μL KAPA HiFi HotStart ReadyMix (2X) per reaction
on ice
See Guidelines section for KAPA primer designKAPA primer stocks should be diluted to
?. [Preparation]
Prepare a diluted SPB Master Mix (SPB MM) +
[SPB]
[nuclease-free water]
?. [Preparation]
Make a 110X mix:  + Vortex to mix
[TB1]
[BLT]
?. [Preparation]
Make a 110X mix:  +  +  + Vortex to mix and briefly spin down
[nuclease-free water]
[KAPA-PCR-F]
[KAPA-PCR-R]
[KAPA HiFi HotStart ReadyMix (2X)]
?. [96-Well Tagmentation]
Transfer at least  (up to 500ng) into each well of a 96-well PCR plate in 1-5μL
[genomic DNA]
?. [96-Well Tagmentation]
Adjust the volume of each well to  using nuclease-free water
[total volume]
?. [96-Well Tagmentation]
Load  into each tube of an 8-strip of PCR tubesVortex TMM thoroughly before use and ensure the beads remain evenly suspended while being aliquoted into the 96-well plate
[TMM]
?. [96-Well Tagmentation]
Add  to each well using an 8-channel pipetteThoroughly mix by gentle pipetting
[TMM]
If beads are adhered to the top or side of the 96-well plate, briefly centrifuge and fully resuspend the bead pellet by pipetting until thoroughly mixed
?. [96-Well Tagmentation]
Seal the plate with a PCR plate cover, place in a thermal cycler, and run the TAGflex program
It is especially important to be able to remove the plate seal as easily as possible to prevent cross contaminationProceed immediately to the next steps upon completion of the TAGflex cycle
?. [Post Tagmentation Cleanup]
Load  per tube in an 8-strip of PCR tubes
[TSB]
?. [Post Tagmentation Cleanup]
Gently remove the plate seal and add  into each tagmentation reaction using an 8-channel pipetteMix by gently pipetting the entire volume, ensuring the beads are fully resuspend
[TSB]
?. [Post Tagmentation Cleanup]
Seal the plate, place in a thermal cycler, and run the TAGstop program
?. [Post Tagmentation Cleanup]
Remove the seal and place the plate on a 96-well magnetic plate stand for  or until solution is clear
?. [Post Tagmentation Cleanup]
Carefully remove the supernatant using an 8-channel pipette without disturbing the beads
?. [Post Tagmentation Cleanup]
Load  into a sterile trough to pipette from
[TWB]
?. [Post Tagmentation Cleanup]
Remove the plate from the magnetic stand and add  to each well using an 8-channel pipetteMix by gently pipetting (slowly, to minimize foaming) until beads are fully resuspended
[TWB]
?. [Post Tagmentation Cleanup]
Place the plate on the magnetic stand for  or until solution is clear
?. [Post Tagmentation Cleanup]
Carefully remove the supernatant using an 8-channel pipette without disturbing the beads
?. [Post Tagmentation Cleanup]
Repeat steps 20-22 one more time for a total of 2 washes
?. [Post Tagmentation Cleanup]
Remove the plate from the magnetic stand and add  to each wellGently pipette until beads are fully resuspended
[TWB]
?. [Post Tagmentation Cleanup]
Seal the plate and place on the magnetic stand to incubate until needed in the next section
Keeping the pellet in TWB helps prevent over-drying of the beads
?. [Amplify Tagmented DNA]
Load  per tube into an 8-strip of PCR tubes (store  until needed)
[PCR MM]
on ice
?. [Amplify Tagmented DNA]
Carefully remove the TWB supernatant from the samples while on the magnetic stand
Any remaining foam on the well walls should not adversely affect the library
?. [Amplify Tagmented DNA]
Remove the plate from the magnetic stand and proceed immediately to the next step to prevent excessive drying of the beads
?. [Amplify Tagmented DNA]
Add  to each sample and pipette the mix to ensure the beads are thoroughly resuspended
[PCR MM]
Pipette with moderate force as the beads can be difficult to fully resuspend
?. [Amplify Tagmented DNA]
Seal the plate and spin down briefly up to
Centrifuge: 1000 33
?. [Amplify Tagmented DNA]
Add  of an equimolar mix of each i7 and i5 index combination to their respective wells
2.5 µl
Individual index tubes: 1.25μL i7 index (N7xx) + 1.25μL i5 index (N5xx)Pre-mixed index plate: Transfer 2.5μL of each index combination using an 8-channel pipette
?. [Amplify Tagmented DNA]
Pipette to mix a minimum of 10 times, then seal the plate and breifly spin up to
Centrifuge: 1000 33
?. [Amplify Tagmented DNA]
Place the plate in a thermal cycler and run the flexPCR program
?. [Amplify Tagmented DNA]
Remove the plate from the thermal cycler and spin down for  at
Centrifuge: 280 34
?. [Amplify Tagmented DNA]
Load  of  per tube into an 8-strip of PCR tubes
35 µl
[proteinase K]
?. [Amplify Tagmented DNA]
Remove the plate seal and add  to each PCR reaction using an 8-channel pipetteMix the wells by pipetting a 10uL volume at least ten times
[proteinase K]
?. [Amplify Tagmented DNA]
Seal the plate, place in a thermal cycler, and run the protK program
?. [Amplify Tagmented DNA]
If stopping, keep the plate sealed and store at 4°C for up to 3 daysOtherwise, continue with the next section
?. [Post-PCR Library Clean Up]
Remove the seal and place the plate on the magnetic stand for  or until the supernatant is clear
?. [Post-PCR Library Clean Up]
Using an 8-channel pipette, transfer  from each well into an 8-strip of PCR tubes
[PCR supernatant]
?. [Post-PCR Library Clean Up]
From the 8-strip of PCR tubes, transfer all samples to a single 1.5mL Eppendorf tube
Each tube in the strip should contain ~24μL of pooled sample, yielding ~192μL of pooled libraries
?. [Post-PCR Library Clean Up]
Vortex and briefly spin down to mix the pooled libraries evenly
?. [Post-PCR Library Clean Up]
Transfer a  of the pooled libraries to a new 1.5mL Eppendorf tube
[aliquot]
?. [Post-PCR Library Clean Up]
Vortex and invert SPB MM multiple times to ensure full resuspension
?. [Post-PCR Library Clean Up]
Add  to the pooled library aliquotPipette until thoroughly mixed
[SPB MM]
Complete mixing is critical to proper size distribution of libraries
?. [Post-PCR Library Clean Up]
Incubate at  for
0 Room temperature
?. [Post-PCR Library Clean Up]
Place the Eppendorf tube on a magnetic stand for  or until the supernatant is clear
?. [Post-PCR Library Clean Up]
Transfer  into a new 1.5mL tube without disturbing the beads
[supernatant]
?. [Post-PCR Library Clean Up]
Vortex and invert the SPB (undiluted stock) until thoroughly resuspended
?. [Post-PCR Library Clean Up]
Add  to the new 1.5mL tube containing the supernatant
[SPB (undiluted stock)]
?. [Post-PCR Library Clean Up]
Pipette a 120μL volume until thoroughly mixed
?. [Post-PCR Library Clean Up]
Incubate at  for
0 Room temperature
?. [Post-PCR Library Clean Up]
Place the Eppendorf tube on a magnetic stand for  or until clear
?. [Post-PCR Library Clean Up]
Remove and discard the supernatant without disturbing the beads
?. [Post-PCR Library Clean Up]
With the tube on the magnetic stand, add  without mixing and incubate for 30s
[80% ethanol]
Add enough 80% ethanol so that the beads are entirely submerged
?. [Post-PCR Library Clean Up]
Pipette to remove the ethanol without disturbing the beads
?. [Post-PCR Library Clean Up]
Repeat steps 55-56 one more time for a total of 2 washes
?. [Post-PCR Library Clean Up]
Remove any excess liquid from the tube using a pipette without disturbing the beads
?. [Post-PCR Library Clean Up]
Air-dry beads on the magnetic stand for
?. [Post-PCR Library Clean Up]
Warm  at  to increase final library yield
[RSB]
50 °C
?. [Post-PCR Library Clean Up]
Remove the 1.5mL tube from the magnetic stand
?. [Post-PCR Library Clean Up]
Add  to the beadsPipette mix until thoroughly resuspended
[RSB (warm)]
?. [Post-PCR Library Clean Up]
Incubate on the bench at   for
0 Room temperature
?. [Post-PCR Library Clean Up]
Place the 1.5mL tube back on the magnetic stand for  or until clear
?. [Post-PCR Library Clean Up]
Transfer  into a new 1.5mL Eppendorf tube
[supernatant]
?. [Post-PCR Library Clean Up]
Let the tube with the eluted library sit at  for  to ensure complete dissolution of the library
50 °C
?. [Post-PCR Library Clean Up]
Quantify the library using a Qubit fluorometerDetermine fragment size distribution using a Bioanalyzer