Introduction
This protocol is a low-cost alternative to the Illumina DNA Prep protocol (previously Nextera DNA Flex). The paper describing Hackflex can be found here. Hack Flex is similar to the official protocol, but uses diluted Bead-Linked Transposomes and lab-made buffers.
The Illumina DNA Prep protocol can be found here.
Reagents
Reagent | Vendor | Catalog | Storage |
Illumina DNA Prep Kit | Illumina | 20060059 | Various |
IDT for Illumina DNA/RNA UD Indexes Set A | Illumina | 20027213 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set B | Illumina | 20027214 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set C | Illumina | 20042666 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set D | Illumina | 20042667 | -20°C |
80% Ethanol, freshly prepared | NA | NA | RT |
Molecular-Grade (Nuclease-Free) Water | Sigma | W4502-1L | RT |
Tris HCI Buffer, pH 7.6, 1 M | Crystalgen | 221-230 | 4°C |
MgCl2, 1 M | VWR | E525-100ML | RT |
N,N-DIMETHYLFORMAMIDE >99%, liquid | Sigma | D4551-250ML | RT, in flammable cabinet |
UltraPure 10% SDS | ThermoFisher | 15553027 | RT |
PrimeSTAR GXL DNA Polymerase Kit | Takara | R050A | -20°C |
Tris-EDTA (TE) Buffer | FisherScientific | BP2473-1 | RT |
SPRI (Homemade) or AmpureXP Beads | 4°C |
Protocol
Prepare Reagents
Bead-Linked Transposomes (BLT) (Stored at 4°C)
Bring to room temperature. Vortex to mix. Do not centrifuge before pipetting.
NEVER put BLT on ice. Do not use BLT stored below 2°C.
(Split between 2 50 mL conicals)
Bring to room temperature. Vortex to mix.
Reagent | [Stock] | [Final] | Volume (ml) |
Tris HCI Buffer, pH 7.6, 1 M | 1 M | 20 mM | 1 |
MgCl, 1 M | 1 M | 20 mM | 1 |
N,N-DIMETHYLFORMAMIDE >99%, liquid | >99% | 50% | 25 |
Molecular Grade Water | NA | NA | 23 |
Total | NA | NA | 50 |
If precipitates are observed, heat at 37°C for 10 minutes and then vortex until dissolved.
Reagent | [Stock] | [Final] | Volume (ml) |
Ultrapure SDS | 10% | 0.2% | 1 |
Molecular Grade Water | NA | NA | 49 |
Total | NA | NA | 50 |
Lab-Made Tagment Wash Buffer (LTWB) (Stored Room Temperature)
Filter through 0.22 um filter before use. Use at room temperature
Reagent | [Stock] | [Final] | Amount |
PEG 8000 | Solid | 10% | 25 g |
NaCl | 5 M | 0.25 M | 12.5 ml |
Tris-EDTA (TE) buffer (10 mM Tris and 1 mM EDTA) | 1X | NA | to 250 ml |
Total | NA | NA | 250 ml |
PrimeSTAR GXL DNA Polymerase kit (Stored -20°C)
Index Adapters (Stored -20°C)
Thaw at room temperature. Spin briefly before use.
Resuspension Buffer (RSB) (Stored 4°C)
Thaw and bring to room temperature. Vortex to mix.
SPRI beads or AmpureXP (Stored 4°C)
Equilibrate to RT for at least 30 min. Vortex and invert to mix.
Tagment DNA
Add 2–10 µl DNA to each well of a 96-well PCR plate so that the total input amount is 10 ng.
If DNA volume < 10 µl, add nuclease-free water to the DNA samples to bring the total volume to 10 µl.
Vortex BLT vigorously for 10 sec to resuspend. Repeat as necessary as beads settle.
Prepare a 1:50 dilution of BLT in molecular-grade water. You will need 11 uL of 1:50 diluted BLT per sample.
Prepare Tagmentation Master Mix (10% overage is already included):
Reagent | Volume | 96 Samples | x Samples |
1:50 BLT | 11 ul | 1,056 ul | |
LTB | 27.5 ul | 2,640 ul |
Vortex the tagmentation master mix thoroughly to resuspend.
Transfer 35 μl tagmentation master mix to each well of the plate containing a sample. If prepping a large number of samples, you may divide the mix volume equally into an 8-tube strip and use a multichannel pippette to transfer.
Pipette each sample 10 times to resuspend.
Seal the plate with Microseal ‘B’, place on the preprogrammed thermal cycler, and run the TAG-FLEX program (55°C for 15 min, hold at 10°C, 45 uL volume and 100°C lid). All programs are under CHMI→NEXTERAFLEX on the thermocycler.
Post-Tagmentation Cleanup
Add 10 µl LTSB to the tagmentation reaction.
Slowly pipette each well 10 times to resuspend the beads.
Seal the plate with Microseal ‘B’, place on the preprogrammed thermal cycler, and run the PTC-FLEX program (37°C for 15 min, hold at 10°C, 55 uL volume, 100°C lid).
After removing from the thermocycler, place the plate on the magnetic stand and wait until liquid is clear (~3 minutes).
Using a multichannel pipette, remove and discard supernatant.
Wash with LTWB twice, as follows:
Remove the sample plate from the magnetic stand and very slowly add 100 µl LTWB directly onto the beads, avoiding foaming.
Pipette slowly until beads are fully resuspended.
Place the plate on the magnetic stand and wait until the liquid is clear (~3 minutes).
Using a multichannel pipette, remove and discard supernatant.
Remove the plate from the magnetic stand and use a slowly pipette to add 100 µl LTWB directly onto the beads.
Pipette each well slowly to resuspend the beads.
Seal the plate and place on the magnetic stand until the liquid is clear (~3 minutes). Keep on the magnetic stand until the appropriate step in the next section.
Amplify Tagmented DNA
Prepare PrimeStar PCR master mix, per sample (overage is already included):
Reagent | Volume (ul) | 96 Samples | X Samples |
5x GXL Buffer | 11 | 1,056 | |
25 mM dNTPs | 4.4 | 422.2 | |
PrimeStar GXL Polymerase | 2.2 | 211.2 | |
Nuclease-free Water | 20.9 | 2,006.4 |
Vortex, and then centrifuge the PCR master mix at 280 × g for 10 seconds.
With the plate on the magnetic stand, remove the seal and use a 200 µl multichannel pipette to remove and discard supernatant (containing TWB). Foam that remains on the well walls does not adversely affect the library.
Remove the plate from the magnet.
Immediately add 35 µl PrimeStar PCR master mix directly onto the beads in each sample well.
Immediately pipette to mix until the beads are fully resuspended. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Seal the sample plate with foil and centrifuge at 280 × g for 3 seconds.
Add 10 ul of the appropriate index adapter to each sample.
Using a pipette set to 35 µl, pipette 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Seal the plate with Microseal ‘B’, and then centrifuge at 280 × g for 30 seconds. Place on the thermal cycler and run the BLT PCR program with a 45 uL volume and 100°C lid:
- 68°C for 3 min
- 98°C for 3 min
- 12 cycles of:
- 98°C for 45 sec
- 62°C for 30 sec
- 68°C for 2 min
- 68°C for 1 min
- 10°C hold
This is a safe stopping point. If you stop here, seal the plate and store at 2°C to 8°C for up to 3 days. Do not leave in the thermal cycler overnight or else samples will evaporate.
Clean Up Libraries
Allow SPRI or Ampure XP Beads to equilibrate to room temperature on the bench for at least 30 minutes before use.
Centrifuge PCR plate at 280 × g for 1 minute to collect contents at the bottom of the well.
Place the plate on the magnetic stand and wait until the liquid is clear (~5 minutes).
Transfer 45 µl supernatant from each well of the PCR plate to the corresponding well of a new deep-well plate.
Due to Hackflex’s lower PCR reaction volume, you may get slightly less supernatant here and in the proceeding steps.
Vortex SPRI or Ampure XP Beads and invert to mix.
Add 45 µl nuclease-free water to each well containing supernatant.
Add 45 µl beads to each well containing supernatant.
Pipette each well 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Seal the plate and incubate at room temperature for 5 minutes.
Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
During incubation, thoroughly vortex SPRI or Ampure XP Beads, and then add 15 µl to each well of a new deep-well plate.
Transfer 125 µl supernatant from each well of the first plate into the corresponding well of the second plate (containing 15 µl SPRI or Ampure XP Beads).
Pipette each well in the second plate 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Discard the first plate.
If your starting material is anything other than gDNA, consult the manual. Smaller input DNA lengths may require a different quantity of beads.
Incubate at room temperature for 5 minutes.
Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
Remove and discard all supernatant from each well without disturbing the beads.
Wash 2 times as follows:
Add 190 μl fresh 80% EtOH to each well.
Incubate on the magnetic stand for 30 seconds.
Remove and discard all supernatant from each well without disturbing the beads.
Using a 20 μl pipette, remove residual 80% EtOH from each well.
Air-dry on the magnetic stand for 5 minutes or until beads just start to dry.
Remove from the magnetic stand.
Add 32 μl RSB to each well.
Pipette to resuspend.
Incubate at room temperature for 2 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Transfer 30 μl supernatant to a new plate.
This is a safe stopping point. If you stop here, seal the plate with foil and store at -20°C for up to 30 days.
Quality Check
- Libraries with DNA Inputs of 100-500 ng generated in the same experiments do not require quantification individually. These can be pooled in a microcentrifuge tube. Add 5 ul of each library (up to 384), vortex to mix, and centrifuge briefly before quantifying the pool.
- Libraries with DNA Inputs of <100 ng require individual quantification.
- Use the HS DNA 5000 or HS DNA 1000 tape and the appropriate reagent buffer. A successful library preparation will have a peak around 500bp. See below for example traces.
- Quantify the sample with the HS DNA Qubit kit.
Standard Nextera Flex Tapestation Trace
HackFlex Tapestation Trace
