TITLE: Assay for determination of functional concentration of Tn5 transposase
AUTHORS: Adrian Mcnairn

[DESCRIPTION]
    Tn5 is used by multiple labs world-wide for its ability to introduce DNA oligos and barcode sequences into libraries and for genomic assays. In many instances, labs produce their own Tn5 enzyme in-house rather than from commercial sources. Our method provides a means of determining the functional concentration of Tn5 in preparations by qPCR to standardize amounts of Tn5 used in assays and identify batch/lot variability. The current standard for assaying in-house produced Tn5 is a plasmid smear. Purified Tn5 is loaded with oligonucleotides and mixed with a plasmid substrate. The plasmid is then run on an agarose gel and checked for smearing, indicating the DNA was cut by the Tn5. The amount of Tn5 is standardized by measuring protein concentration using a protein assay or absorbance at 280nM which provides an approximation based on total protein present, not the functional concentration.This method is modified from Rykalina et al. to evaluate and empirically determine the functional concentration of Tn5 transposomes in homemade enzyme preps by qPCR. The principle is based on decreased Cp(or Cq) values correlating with increased fragmentation caused by the transposome.The change in Cp values functions as a measurement of the activity of the Tn5 at that concentration of oligonucleotide (the greater the number of cycles required to produce a product correlates with the increased cleavage of the plasmid substrate by Tn5) as plasmids with transposomes insertions (cleaved regions) will not amplify. The change in Cp values is then plotted against the oligonucleotide concentration in a line graph and a plateau will appear at the concentration at which the Tn5 is saturated by oligo. The first point in the plateau is the functional concentration.
    This method may also be applied to testing the efficiency of changing DNA sequences in oligos used to assemble transposomes as the activity of the transposase is dependent upon the binding sequences contained within the oligo as well as secondary structure formed by the oligo. This property enables testing of oligo variations and barcode efficiencies in our assay. For instance, oligos containing different lengths or different barcodes sequences can be assembled in transposomes and tested in comparison to standard oligos

[STEPS]
SECTION: Transposome preparation and assembly
1. Prepare ME A/Rev or ME-B/rev oligos by annealing equal concentrations of primers. 
We typically target 40uM stocks.
 Example: 40uL 100uM Tn5-ME-A +40uL 100uM Tn5-REV +20uL nuclease-free water or 0.1x TE (can be scaled as needed)
Thermocycler program: 95°C for 2min, slow cool to 25deg at 0.1°C/sec, total time ~22 minutes. 22 min  

SECTION: Transposome preparation and assembly
2. Prepare Tn5 transposomes by combining 9uL of the Tn5 to be tested with 1uL annealed oligos (equates to final concentration of: 0.125uM,0.25uM, 0.5uM, 1uM, 2uM, 4uM)
a.  If the estimated concentration of Tn5 is high, prepare serial dilutions first in Tn5 exchange buffer
b.  Volumes may be titrated down if Tn5 to be tested is in limited quantities(i.e 0.5uL oligo in 4.5uL Tn5) 
   

SECTION: Template preparation
3. 60 min                                                                                                                                                                                                                                                                                                                                                                                                                     Digest pUC19 with EcoRI to generate a linear template. 
a. Column purify the plasmid DNA and adjust concentration to 25ng/uL
b. The digest reaction may be scaled to provide a stock of linearized plasmid for future use
SECTION: Tagmentation
4. In duplicate, prepare tagmentation master mix and test each concentration of transposome on 25ng linearized pUC19
a.  Include a no Tn5 control for reference (0uM) 
b.  For no Tn5 control, increase water to 11.5uL/reaction
c. 10X Tango restriction enzyme buffer (ThermoFisher) or 10X Cutsmart buffer(NEB) are used to provide the magnesium Tn5 requires for tagmentation. A recipe for an alternative 2X buffer is listed below. 
 
StockVol per 25uL rxn (uL)10X Tango RE Buffer2.5Nuclease-free water18DMF2.5Linearized pUC19(25ng/uL)1Tn51
 2X Tagmentation Buffer
 
StockVol for 10mLFinal Conc. Supplier1M Tris-HCl pH7-8200uL20mMInvitrogen #AM9850G, #AM9855G1M MgCl2100uL10mMInvitrogen #AM9530GNuclease-free water9.7mL Invitrogen AM9932
 
SECTION: Tagmentation
5. Incubate at 37°C for 1hr at 600rpm
SECTION: Tagmentation
6. Stop the reaction by adding 1uL of 2%SDS to each reaction(final conc. 0.08%)
a.    55°C for 7 minutes to remove transposomes7 min 

SECTION: Tagmentation
7. Quench SDS by adding 3ul 10% Triton X-100 to reactions
SECTION: Tagmentation
8. Final volume should be 29uL
SECTION: Tagmentation
9. Qubit or nanodrop DNA for normalization (~0.86ng/uL)
a.    Quantifying the DNA first enables the assay to be more quantitative
SECTION: qPCR
10. Dilute transposed DNA 1:10 in nuclease free water for qPCR
a.   using too much DNA may lead to difficulty in determining Cp and lead to higher data variability
b.   Controls: linearized pUC19 without Tn5 and a NTC control
SECTION: qPCR
11. Setup qPCR reactions using primer pairs (recommend running at least 2 pairs)
a.    Pairs 591, 602, and 610 may be run on the same program

SECTION: qPCR
12. Run qPCR using following settings:
a.    95°C for 1 min, followed by 35(minimum) to 45 cycles of 95°C for 15 sec, 60°C for 20 sec, 72°C for 30sec, then a melt curve
Two-step protocols may also be used alternating 95°C for 15 sec, 60°C for 20 sec
SECTION: qPCR
13. Analyze and graph delta Cp versus concentration. 
a. The no Tn5 control provides the reference value as the plasmid should be intact (i.e Subtract the Geomean of the no Tn5 replicates from the Cp of the test samples). 
b. The higher the delta Cp, the more cleavage of the plasmid, indicating higher Tn5 tagmentation activity
c. The concentration at which the graph plateaus or peaks at, is the functional concentration of the Tn5 prep

SECTION: Transposome preparation and assembly
1.1. Prepare working stocks of annealed oligos by serial dilution(1:1):  1.25uM, 2.5uM, 5uM, 10uM, 20uM, 40uM
SECTION: Transposome preparation and assembly
2.1.  
 Oligo Concentration(uM)1.252.55102040Oligo(uL)111111Tn5(uL)999999Final vol101010101010Final Conc.(uM) 0.1250.250.5124
 
SECTION: Transposome preparation and assembly
2.2.  Incubate for 21 hours at 25°C w/shaking(600rpm) on Eppendorf Thermomixer   
Shorter incubations (1 hour) are possible, but we find the longer incubations provides better, more consistent, results. 
SECTION: Data Analysis Example
14.  
 Primer pair:610SampleCpAve Cpdelta Cp0.58.898.788.988.880.740.59.128.818.978.970.8319.749.89.749.761.6219.679.999.799.821.682.211.3611.3411.4911.403.262.211.3711.6711.5711.543.403.512.5212.6312.7312.634.493.512.712.7412.8612.774.6359.839.949.929.901.7659.7910.059.979.941.80noTn58.28.028.188.13-0.01noTn58.257.978.218.140.00NTC24.0423.9223.923.9515.81NTC24.2724.2524.2624.2616.12
  
 Primer pair:610Conc.(uM)ave delta CPstdev0.5uM0.790.061uM1.650.042.2uM3.330.103.5uM4.560.105uM1.780.03