Titration of Tn5 transposition in MEFs

Part 1. Assembling the Transposase

1. Resuspend primers to 100uM stock using Annealing Buffer (40mM Tris-HCl (pH8.0), 50mM NaCl)

2. Mix the following :

   o Primer A + Mosaic Rev (10uL + 10uL)

   o Primer B + Mosaic rev (10uL + 10uL)

3. Anneal using the following settings:

Temperature	Time
95°C	5 minutes
Cool to 65°C	-0.1°C/second
65°C	5 minutes
Cool to 4°C	-0.1°C/second

4. In a PCR tube, mix the Following (scaled just enough for small scale titration + 10%).

Components	Diagenode Tn5	InHouse
Primer A-Mosaic	1uL	2.5uL
Primer B-Mosaic	1uL	2.5uL
Tn5	2uL	5uL

5. Vortex briefly and incubate at 23°C for 30 minutes in a thermocycler.

Part 1.1 Prepping the Tn5 concentrations from Assembled Tn5 transposome per Transposition reaction (50uL)

1. Prep 3 tubes for each Tn5 variant, one for each concentration to be tested.

2. Follow the example Transposition mix composition below good for 20 rxns, scale according to # of transposition reactions. Adjust Tn5 volume for desired concentration to be tested.

Component	Volume
2X TD buffer (recipe below)	500uL
PBS	330uL
UltraPure dH2O	100uL
1% Digitonin	10uL
10% Tween-20	10uL
Assembled Tn5 (diluted)	20uL
Total	1mL

Recipe for 2X TD buffer

Component	Final Concentration	Volume for 100mL
1M Tris-HCl pH 7.6	20 mM	2 ml
1M MgCl2	10 mM	1 ml
Dimethyl Formamide	20%	20 ml
Sterile water	NA	Bring up to 100 ml

Part 2. Transposition Reaction of Isolated nuclei

1. Resuspend the isolated nuclear pellet in 50 µL of Transposition Mix (Pre-made from Part 1.1) by pipetting up and down six times. Transposition Mix should be made fresh each time and mixed thoroughly prior to use.

2. Incubate reaction at 37 °C for 30 min in a thermomixer with 1,000 rpm mixing.

3. Remove the tubes from the thermomixer, and immediately terminate the transposition reaction by adding 250 µL (five volumes) of DNA Binding Buffer from the DNA Clean and Concentrator-5 Kit and mix well by pipetting or inversion.

4. Pulse centrifuge to collect solution in the bottom of the tube.

5. Clean up the transposition reaction using the DNA Clean and Concentrator-5 Kit. Transfer each sample, mixed with the DNA Binding Buffer, to a Zymo-Spin Column in a collection tube. Centrifuge at RT for 30 s at 10,000g, and discard the flowthrough.

6. Add 200 µL of DNA Wash Buffer to the column, and centrifuge at RT for 30 s at 10,000g.

7. Repeat this wash for a total of two wash steps.

8. Perform a final ‘dry spin’ after the second wash step to remove any traces of residual wash buffer from the column membrane. To do this, remove any flowthrough from the collection tube and centrifuge the column and collection tube at RT for 1 min at >13,000g.

9. Transfer the column to a clean prelabeled 1.5 mL LoBind tube. Pipette 21 µL of Elution Buffer directly onto the column membrane, and wait for 1 min.

10. Centrifuge the column at RT for 1 min at 13,000g to elute the DNA. This elution volume typically results in 20 µL of product.

Pause Point: This can be Stored at -20C for as long as necessary or processed immediately for Barcode amplification followed by sequencing.