Mar 25, 2024
Plate Scale Tn5 based tagmentation library prep protocol
- Cade Mirchandani1,2,
- max genetti1,2,
- Pingting Wang1,
- Evan Pepper-Tunick3,4,
- Shelbi Russell1,2,
- Russell Corbett-Detig1,2
- 1Department of Biomolecular Engineering, University of California, Santa Cruz;
- 2Genomics Institute, University of California Santa Cruz, Santa Cruz, CA, United States;
- 3Institute for Systems Biology, Seattle, Washington, USA;
- 4Molecular Engineering and Sciences Institute, University of Washington, Seattle, Washington, USA
Protocol Citation: Cade Mirchandani, max genetti, Pingting Wang, Evan Pepper-Tunick, Shelbi Russell, Russell Corbett-Detig 2024. Plate Scale Tn5 based tagmentation library prep protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qmzpl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 24, 2024
Last Modified: March 25, 2024
Protocol Integer ID: 97191
Keywords: Tn5 based tagmentation library, Transposon Assembly, PCR Enrichment, Gel extraction, Size selection, Plate scale
Funders Acknowledgements:
NIH
Grant ID: R35GM128932
NIH
Grant ID: R00GM135583
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Abstract
This protocol is a fork of (dx.doi.org/10.17504/protocols.io.bv5gn83w), which a demonstrated efficient and high-throughput tagmentation sequencing library prep protocol based on Picelli et al 2014. In this version, the tagmentation and amplification steps remain largely the same - based on the Tn5 transposase and KAPA HiFi kit, respectively. Here, significant modifications have been made to the library pooling and cleanup processes. Specifically, this protocol involves pooling the crude PCR products, followed by the removal of small fragments via a spin column kit, and depletion of large fragments through agarose gel extraction. These modifications enable faster processing times while still producing high quality sequencing libraries.
This protocol has been optimized for sequencing Drosophila cell culture samples, from which high-quality genomic DNA (gDNA) can be readily obtained. The accessibility to high-quality gDNA simplifies the normalization of gDNA input amounts prior to tagmentation, thereby streamlining the pooling and cleanup steps.
Guidelines
Appendix 1
Oligo Sequences:
| A | B | C | |
| Name | Sequence | Concentration | |
| Tn5ME-R | 5'-[phos]CTGTCTCTTATACACATCT-3' | 100uM | |
| Tn5ME-A | 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’ | 100uM | |
| Tn5ME-B | 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’ | 100uM |
Index Primers:
PCR index primer are following Nextera XT Index Kit v2 - Index 2 (i5/i7) Adapters, sequences can be found on page 14 of Illumina adapter sequences. You can either order the whole kit from Illumina or synthesize it your self. I was using orders from IDT with standard desalting and it worked fine.
Materials
Tagmentation:
Tn5 Enzyme Mix:
| A | B | |
| Tn5 | 0.2-0.4 mg/mL | |
| Tris-HCl, pH 7.5 | 25 mM | |
| NaCl | 800 mM | |
| EDTA | 0.1 mM | |
| DTT | 1 mM | |
| Glycerol | 50% |
5X TAPS-PEG 8000:
50 mM TAPS-NaOH at pH 8.5, 25 mM MgCl2,, PEG 8000 40%
PCR Amplification:
- KAPA HiFi PCR Kit (KAPA code KK2101; Roche Catalog #07958838001)
Size Selection:
- ZYMO Select-A-Size DNA Clean and Concentrator Kit (Catalog #D4080)
- NEB Monarch DNA Gel Extraction Kit (Catalog #T1020S)
- NEB 6X gel loading dye (Catalog #B7024S)
- NEB 100bp DNA ladder (Catalog #N3231S)
- Invitrogen SYBR Gold (Catalog #S11494)
QC
- Thermofisher Qubit dsDNA Quantitation, High Sensitivity (Catalog #Q32851)
- Agilent D1000 ScreenTape (Catalog #5067-5582)
- Agilent D1000 Reagents (Catalog #5067-5583)
Before start
- AR and BR oligo aliquots can be prepared in advance by combining equal volumes of Tn5ME-A/B and Tn5ME-R. Store at -20C. To use, thaw on bench, vortex and quick spin, then proceed to step 3. This is recommended to minimize freeze/thaw cycles on oligo stocks.
- A normalized gDNA plate should be prepared before starting. 10-20ng/ul is sufficient.
Transposon Assembly
Transposon Assembly
2h 1m
2h 1m
Remove oligos Tn5ME-A, Tn5ME-B, Tn5ME-R, and Tn5 enzyme mix from freezer. Thaw primers, mix by vortexing and spin. Keep Tn5 enzyme On ice . Turn on 95 °C thermocycler.
In separate PCR tubes combine:
7 µL Tn5ME-A + 7 µL Tn5ME-R = AR oligo (14 µL )
7 µL Tn5ME-B + 7 µL Tn5ME-R = BR oligo (14 µL )
Place oligos in thermocycler on the following program:
1. 95 °C x 00:05:00
2. 25 °C x -0.1 °C /sec
3. 4 °C x ∞
5m
In separate 1.5 mL tubes combine the following:
13 µL AR oligo + 91 µL Tn5 = Tn5-AR (104 µL )
13 µL BR oligo + 91 µL Tn5 = Tn5-BR (104 µL )
Mix by pipetting gently.
5m
Incubate at Room temperature for 01:00:00 .
1h
Tagmentation
Tagmentation
2h 17m
2h 17m
Preheat thermocycler to 55 °C
Prepare tagmentation plate by aliquoting 2 µL (10-20 ng ) gDNA to each well of new 96 well plate.
5m
In new 2 mL tube prepare Tn5 Reaction Master Mix:
| A | B | C | |
| Reagent | Volume (uL) x 100rxns | Volume (uL) x 1 rxn | |
| Molecular grade water | 1200 | 12 | |
| 5x TAPS-PEG 8000 | 400 | 4 | |
| Tn5-AR | 100 | 1 | |
| Tn5-BR | 100 | 1 | |
| Total | 1800 | 18 |
Mix by inverting.
Aliquot 18 µL of master mix to each well of tagmentation plate. Seal with foil and spin.
5m
- Incubate in thermocycler 00:08:00 at 55 °C .
- Remove plate from thermocycler and immediately place On ice .
8m
- Carefully remove film and add 5 µL 0.2% SDS (prepared in a strip tube).
- Seal with new film and incubate for 00:10:00 at Room temperature
10m
PCR Enrichment
PCR Enrichment
7m
7m
Remove KAPA HiFi dNTPs and Fidelity 5X buffer from freezer and thaw, vortex, and spin. Index primer plates should be thawed and spun down.
Note
KAPA HiFi PCR enzyme should only be out of the freezer briefly.
In a 2 mL tube prepare PCR Master Mix
| A | B | C | |
| Reagent | Volume (uL) x 100rxns | Volume (uL) x 1 rxn | |
| Molecular grade water | 1175 | 11.75 | |
| 5X Fidelity Buffer | 500 | 5 | |
| 10 mM dNTPs | 75 | 0.75 | |
| HiFi | 50 | 0.5 | |
| Total | 1800 | 18 |
Mix by inverting and spin. Store On ice .
5m
Prepare PCR plate by adding the following to each well in new 96 well plate:
- 2 µL combined index primers (1 µL 5uM forward & 1 µL 5uM reverse).
- 5 µL tagmentation product from tagmentation plate.
- 18 µL PCR Master Mix
Note
Be careful that plate positions match. Make sure to record index primers used.
5m
Seal PCR plate with foil, spin, and place in thermocyclers for the following program:
| A | B | C | |
| Temp (°C) | Time | Cycles | |
| 72 | 5:00 | - | |
| 95 | 3:00 | - | |
| 98 | 0:20 | 12 | |
| 65 | 0:15 | ||
| 72 | 0:30 | ||
| 72 | 5:00 | - | |
| 4 | ∞ | - |
45m
Check concentration of 8-10 randomly selected wells. Desired concentration on Qubit is 15-75ng/uL.
10m
When complete freeze at -20 °C Overnight or proceed to cleanup.
7m
Pooling and Left-side Size Selection
Pooling and Left-side Size Selection
1h 34m
1h 34m
In a 1.5 mL tube, add 2.5 µL from each well of PCR plate, total 240 µL crude pooled library. Prepare ZYMO Select-A-Size DNA Clean and Concentrator Kit.
5m
In order to not exceed the capacity of the Zymo-Spin IC-S column, split the pool into two replicates:
In 2 new 1.5 mL microcentrifuge tubes add the following:
- 100 µL crude pooled library
- 500 µL Select-a-Size DNA Binding Buffer
Mix thoroughly by pipetting the entire volume up and down 5 times
2m
Steps 19.1 - 19.4 are to be done for each prepared binding mixture.
Transfer the binding mixture to a Zymo-Spin IC-S Column in a Collection Tube. Centrifuge at 10,000 x g for 00:00:30 . Discard the flow-through.
30s
Add 700 µL of DNA Wash Buffer to the column. Centrifuge at 10,000 x g for 00:00:30 . Discard the flow-through.
30s
Add 200 µL of DNA Wash Buffer to the column. Centrifuge at 10,000 x g for 00:01:00 . Discard the Collection Tube.
1m
Transfer the column to a new 1.5 mL tube, add 22 µL (This number can be varied based on the concentration of the pooled library) of DNA Elution Buffer directly to the column matrix, and incubate for 00:01:00 at Room temperature . Centrifuge at 10,000 x g for 00:00:30
1m 30s
Check and record concentration of purified pool replicate on Qubit.
5m
Purified pools can be stored at -20 °C .
Right-side Size Selection via Gel Extraction and Final QC
Right-side Size Selection via Gel Extraction and Final QC
2h 20m
2h 20m
- Cast a 1.2% agarose gel (TBE; 0.5cm thick).
- While gel is solidifying, prepare:
- NEB 6X gel loading dye
- NEB 100bp DNA ladder
- NEB Monarch DNA Gel Extraction Kit
3. Thaw purified pools on bench and quick spin.
Note
Ensure you add the appropriate amount of nucleic acid gel stain via pre-loading, precasting, or post-staining.
40m
To prevent overloading gel lanes and minimize gel thickness, divide each pool replicate between two PCR tubes.
- Aliquot 10 µL from each pool replicate into two new PCR tubes, resulting in a total of 4 PCR tubes.
- To each tube, add 2 µL 6X gel loading dye.
2m
In a new PCR tube, prepare DNA ladder by combining:
- 4 µL Molecular biology grade water
- 1 µL 100bp DNA Ladder
- 1 µL 6X gel loading dye
2m
- Load each pool and ladder on agarose gel
- Run gel at 85 V for approx. 01:00:00
1h
Prepare 4 1.5 mL microcentrifuge tubes. Label each, and record their weights.
5m
- Extract gel slices from lanes the pools were run on. Desired size range is between ~450-800bp.
- Place extracted slice into new, labeled 1.5 mL microcentrifuge tube.
10m
Re-weigh microcentrifuge tubes with gel slices and record difference to find gel slice weight.
5m
Steps 28.1 - 28.6 are to be done for each extracted gel slice.
If the gel slice is greater than 150 mg , add 3 volumes of Gel Dissolving Buffer to the gel slice. Else, add 4 volumes.
2m
Incubate at 50 °C for approx. 00:10:00 , inverting periodically until the gel slice is completely dissolved.
10m
- Insert the column into the collection tube and load the sample onto the column.
- Spin at 16,000 x g for 00:01:00
- Discard the flow-through
1m
- Re-insert column into collection tube.
- Add 200 µL DNA Wash Buffer
- Spin at 16,000 x g 00:01:00
- Discard the flow-through.
Repeat once.
1m
Transfer column to a new 1.5 mL microcentrifuge tube.
- Add 16 µL of DNA Elution Buffer to the center of the matrix.
- Incubate for 00:01:00 at Room temperature
- Spin at 16,000 x g for 00:01:00 to elute DNA.
2m
Pool all 4 elutions in to a new 1.5 mL tube. This is the final pool.
Check final pool on Qubit and Agilent Tapestation.
Protocol references
Picelli S, Björklund AK, Reinius B, Sagasser S, Winberg G, Sandberg R. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 2014 Dec;24(12):2033-40. doi: 10.1101/gr.177881.114. Epub 2014 Jul 30. PMID: 25079858; PMCID: PMC4248319.