Hack Flex - Zephyr

Hack Flex - Zephyr

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
Plasticware
Vendor
Catalog
96-well Hard Shell Plates (HSP) for Zephyr
REVVITY
6008870
150 ul filter tips for Zephyr
REVVITY
111426
StorPlate 450 ul V bottom plates for Zephyr
REVVITY
6008290
2 ml Deep Well Plate (DWP) for Zephyr
REVVITY
6008880
Lids for Zephyr
REVVITY
6000030
12-Column Reservoir Seahorse DWP for Zephyr
REVVITY
6008700
287 ml Reservoir for Zephyr
REVVITY
6008730

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.
Lab-Made Tagmentation Buffer 1 (LTB) (Stored -20°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
Lab-Made Tagment Stop Buffer (LTSB) (Stored Room Temperature)

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.

Dilute DNA

❗
This should be done immediately before library prep, and no more than 24 hours in advance.
Create an Excel document to dilute your extracted DNA to 1 ng/ul. The total volume (DNA + EB/Water) needs to be between 12 and 170 ul. This is a snippet of the Excel document that needs to be uploaded to the Zephyr computer in C:\Pooling:
Normalization and Pooling
Sample Well ID
Sample Volume
Diluent Volume
Destination Well ID
A01
5
148
A01
B01
5
76
B01
C01
9
4.23
C01
D01
5
19
D01
E01
5
149.5
E01
F01
5
156.5
F01
…
…
…
…
Open Maestro Workstation and open the “Pooling and Normalization” protocol:

(File → Open Application → Pooling and Normalization).

Push the green Start arrow.
Load deck as follows:
Item
Position
Plasticware
Contents
150 ul Filter Tips
A2
Box Filter Tips
Full Box
DNA Source Plate
A3
96-well HSP
Extracted DNA (thawed, vortexed and spun down)
DNA Destination Plate
B3
96-well HSP
Empty
EB/Water
B4
287 ml Reservoir
EB or Water or C6
Grease the 96-well plate head (only 1x per day).
Press the Start button.
Open the input Excel file (located in C:\Pooling).
Press the Continue button. The program takes about 90 minutes.
Once the program finishes, cover the DNA dilution plate with foil, vortex, and spin down.
Cover the DNA source plate in foil and return to the -20C freezer.

Run the “Nextera Flex Tagmentation and PCR Setup” Protocol on Zephyr

Open Maestro Workstation and open the “HackFlex - DNA Prep No Wash” protocol:

(File → Open Application → Illumina Flex - DNA Prep).

Push the green Start arrow.
Select the appropriate number of columns and then click the “Nextera Flex Tagmentation and PCR Setup” button on the popup.
Load the deck as follows using the prompts:
❗
All volumes are for a full 96-well plate. Adjust as necessary using the HackFlex Zephyr Workbook.xls document (C:\ProgramData\CaliperLS\Maestro\Workbooks).
Item
Position
Plasticware
Contents
150 ul Filter Tips
A1, A2, B1, B2
Box Filter Tips
Full box
BLT/LTB Broadcasting Plate
A3
96-well HSP
Empty
LTSB Broadcasting Plate
A3
96-well HSP
Empty
Waste Reservoir
B4
96-well DWP
Empty
Reagent Plate
C2 on CPAC adapter
StorPlate 450 ul V-bottom with Lid
Column 1: 1:50 BLT/LTB Mix (477 ul per well)        * BLT: 22.4 ul        * H2O: 1098 ul (2x 549 ul) * LTB: 2800 ul Column 3: LTSB (137 ul per well) Column 5: PrimeStar PCR Mix (244 ul per well)        * 5x GXL Buffer: 1244 ul (2x 622 ul)        * dNTPs: 498 ul        * Polymerase: 249 ul
Diluted DNA Plate
C3 on magnet
96-well HSP
10 ul per well of sample DNA at 1 ng/ul
i5/i7 Index Primer Plate
B3 (after prompt)
96-well HSP
10 ul per well of index from IDT for Illumina DNA/RNA UD Index plate
Water Plate
A3 (after prompt)
96-well HSP
75 ul per well
Press the Finish button to start the run. The program takes about 80 minutes.
When prompted (after ~55 minutes) add the i5/i7 Index Primer and Water Plates.
When prompted (after ~70 minutes) add more 150 ul filter tips.
When the protocol finishes, seal the plate with Microseal ‘B’, and 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 -20°C for up to 3 days. Do not leave in the thermal cycler overnight or else the samples will evaporate.

Run the “Standard Post-PCR Size Selection” Protocol on Zephyr

❗
This protocol will automatically start after the previous protocol as long as the application isn’t closed. If it was closed, restart the application, select the appropriate number of columns, and then press the “Standard Post-PCR Size Selection” button.
Load the deck as follows using the prompts:
❗
All volumes are for a full 96-well plate. Adjust as necessary using the HackFlex Zephyr Workbook.xls document (C:\ProgramData\CaliperLS\Maestro\Workbooks).
Item
Position
Plasticware
Contents
150 ul Filter Tips
A1, A2, B1, B2
Box Filter Tips
Full box
Diluted SPB Plate
A3
96-well HSP
Empty (stacked)
Undiluted SPB Plate
A3
96-well HSP
Empty (stacked)
SPB Plate
B3 (on shaker)
96-well HSP
57 ul per well of IBP/AmpureXP/SPRI beads
Waste Reservoir
B4
2 ml DWP
Empty
RSB Plate
C2 on CPAC adapter
96-well HSP with Lid
32 ul per well of RSB or Water THIS WILL BE THE FINAL ELUTION PLATE
Water Plate
C3 on magnet
96-well HSP
Saved from previous protocol.
Ethanol Plate
C4
12-column Reservoir Seahorse Deepwell
4 ml per well of 80% Ethanol (freshly made)
Sample Plate
(after prompt)
96-well HSP
From PCR Machine. Spin down before loading.
Press the Finish button to start the run. The program takes about 60 minutes.
When prompted (~5 minutes), add the Sample Plate.
When prompted (~50 minutes), add more 150 ul filter tips.
🛑
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.

Next Steps

  • 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 (i.e. HackFlex libraries!) require individual quantification.
  • Use the HS DNA 5000 tape and the appropriate reagent buffer and ladder. A successful library preparation will have a peak of around 500 bp. See below for example traces. Calculate the average fragment size of the individual libraries.
  • Quantify the libraries with the Picogreen.
  • Use the concentration and average fragment size to calculate the molarity of individual samples.
  • Pool equal molar concentrations of each library.
  • Calculate pool molarity by measuring concentration via Qubit and average fragment size via TapeStation.
  • Dilute to 2 nM (NextSeq) or 1 nM (MiniSeq) before preparing the run

HackFlex Tapestation Trace