TITLE: Plate Scale Tn5 based tagmentation library prep protocol
AUTHORS: Cade Mirchandani, max genetti, Pingting Wang, Evan Pepper-Tunick, Shelbi Russell, Russell Corbett-Detig

[DESCRIPTION]
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.

[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. 

[GUIDELINES]
Appendix 1

 Oligo Sequences:
 

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.


[STEPS]
SECTION: Transposon Assembly
1. 

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.

SECTION: Transposon Assembly
2. 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)

SECTION: Transposon Assembly
3. Place oligos in thermocycler on the following program:

1. 95 °C x 5 min
2. 25 °C x -0.1 °C /sec
3. 4 °C   x ∞




SECTION: Transposon Assembly
4. 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.
SECTION: Transposon Assembly
5. Incubate at Room temperature for 60 min.

SECTION: Tagmentation
6. Preheat thermocycler to 55 °C 

SECTION: Tagmentation
8. In new 2 mL tube prepare Tn5 Reaction Master Mix:
        900uL H2O  4.5uL    400uL 5x TAPS buffer  2uL    400uL 40% PEG  2uL    200uL Tn5 mix  1uL    DNA (10ng/uL)  1uL    1900uL mix    
Mix by inverting. 
Aliquot 18 µL of master mix to each well of tagmentation plate. Seal with foil and spin.

SECTION: Tagmentation
9. Incubate in thermocycler 8 min at 55 °C. 
Remove plate from thermocycler and immediately place on ice .

SECTION: Tagmentation
10. Carefully remove film and add 5 µL 0.2% SDS (prepared in a strip tube). 
Seal with new film and incubate for 10 min at Room temperature 

SECTION: PCR Enrichment
11. Remove KAPA HiFi dNTPs and Fidelity 5X buffer from freezer and thaw, vortex, and spin. Index primer plates should be thawed and spun down.
 

SECTION: PCR Enrichment
12. In a 2 mL tube prepare PCR Master Mix
 
 
 Mix by inverting and spin. Store on ice .

SECTION: PCR Enrichment
13. 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

 



SECTION: PCR Enrichment
14. Seal PCR plate with foil, spin, and place in thermocyclers for the following program:
 


SECTION: PCR Enrichment
16. When complete freeze at -20 °C 7 min or proceed to cleanup.

SECTION: Pooling and Left-side Size Selection
17. 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.

SECTION: Tagmentation
7. Prepare tagmentation plate by aliquoting 2 µL  (10-20 ng) gDNA to each well of new 96 well plate. 

SECTION: PCR Enrichment
15. Check concentration of 8-10 randomly selected wells. Desired concentration on Qubit is 15-75ng/uL.
SECTION: Pooling and Left-side Size Selection
19.2. Add 700 µL of DNA Wash Buffer to the column. Centrifuge at 10,000 x g for 30 s. Discard the flow-through.

SECTION: Pooling and Left-side Size Selection
19.3. Add 200 µL of DNA Wash Buffer to the column. Centrifuge at 10,000 x g for 1 min . Discard the Collection Tube.

SECTION: Pooling and Left-side Size Selection
19.4. 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 1 min at Room temperature. Centrifuge at 10,000 x g for 30 s
SECTION: Pooling and Left-side Size Selection
19. Steps 19.1 - 19.4 are to be done for each prepared binding mixture.
SECTION: Pooling and Left-side Size Selection
18. 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
SECTION: Pooling and Left-side Size Selection
19.1. Transfer the binding mixture to a Zymo-Spin IC-S Column in a Collection Tube. Centrifuge at 10,000 x g for 30 s. Discard the flow-through.

SECTION: Pooling and Left-side Size Selection
19.5. Check and record concentration of purified pool replicate on Qubit.
SECTION: Pooling and Left-side Size Selection
20. Purified pools can be stored at -20 °C.

SECTION: Right-side Size Selection via Gel Extraction and Final QC
21. 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. 
 


SECTION: Right-side Size Selection via Gel Extraction and Final QC
22. 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. 

SECTION: Right-side Size Selection via Gel Extraction and Final QC
23. 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


SECTION: Right-side Size Selection via Gel Extraction and Final QC
24. Load each pool and ladder on agarose gel 
Run gel at 85 V for approx. 60 min 


SECTION: Right-side Size Selection via Gel Extraction and Final QC
25. Prepare 4 1.5 mL microcentrifuge tubes. Label each, and record their weights.

SECTION: Right-side Size Selection via Gel Extraction and Final QC
26. 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.
SECTION: Right-side Size Selection via Gel Extraction and Final QC
27. Re-weigh microcentrifuge tubes with gel slices and record difference to find gel slice weight.
SECTION: Right-side Size Selection via Gel Extraction and Final QC
28. Steps 28.1 - 28.6 are to be done for each extracted gel slice.
SECTION: Right-side Size Selection via Gel Extraction and Final QC
28.1. If the gel slice is greater than 150 mg , add 3 volumes of Gel Dissolving Buffer to the gel slice. Else, add 4 volumes.

SECTION: Right-side Size Selection via Gel Extraction and Final QC
28.2. Incubate at 50 °C  for approx. 10 min , inverting periodically until the gel slice is completely dissolved. 

SECTION: Right-side Size Selection via Gel Extraction and Final QC
28.3. Insert the column into the collection tube and load the sample onto the column. 
Spin at 16,000 x g for 1 min 
Discard the flow-through

SECTION: Right-side Size Selection via Gel Extraction and Final QC
28.4. Re-insert column into collection tube. 
Add 200 µL DNA Wash Buffer 
Spin at 16,000 x g 1 min
Discard the flow-through. 
Repeat once.

SECTION: Right-side Size Selection via Gel Extraction and Final QC
28.5. Transfer column to a new 1.5 mL microcentrifuge tube.
SECTION: Right-side Size Selection via Gel Extraction and Final QC
28.6. Add 16 µL of DNA Elution Buffer to the center of the matrix. 
Incubate for 1 min  at Room temperature
Spin at 16,000 x g for 1 min  to elute DNA.

SECTION: Right-side Size Selection via Gel Extraction and Final QC
29. Pool all 4 elutions in to a new 1.5 mL tube. This is the final pool.

SECTION: Right-side Size Selection via Gel Extraction and Final QC
30. Check final pool on Qubit and Agilent Tapestation.