ATAC-see
Warning
ATAC-see is a very inefficient procedure, and gives effectively useless results compared to background, even in flow cytometry.
Transposase buffer preparation
Estimated time
1.5 hours.
Order the following primers:
Primer name
Scale
Purification
5’ modification
Sequence
Tn5ME_rev
25nmol
DSL
Phosphate
CTGTCTCTTATACACATCT
Tn5ME_A
50nmol
HPLC
Cy5
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
Tn5ME_B
50nmol
HPLC
Cy5
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
Resuspend each primer separately to 100 uM (the standard stock dilution, nmol * 10 uL).
Prepare 1:1 solutions of Tn5ME_rev / Tn5ME_A (A+rev) and Tn5ME_rev / Tn5ME_B (B+rev). Each primer is now at 50 uM.
Assemble these double stranded oligos by heating to 95C for 5 minutes, followed by slow cooling to room temperature. To do this on a thermocycler, use a program that does ends after the denaturation step, instead of ending in a hold step.
Prepare 5 uM stock solution of Tn5/Cy5. Prepare this either from freshly purified Tn5 in dialysis buffer:
Component
Volume fraction
A+rev primers
0.125
B+rev primers
0.125
Glycerol
0.4
Dialysis buffer
0.12
50 uM Tn5
0.1
DI H2O
0.13
If Tn5 is already stored in a 50% glycerol/50% 2x dialysis stock solution at 20 uM, use the following recipe:
Component
Volume fraction
A+rev primers
0.125
B+rev primers
0.125
Glycerol
0.25
20 uM Tn5+Glycerol
0.25
DI H2O
0.25
ATAC-see
We need the following prepared buffers:
Nuclear permeabilization buffer:
Component
Concentration
Amount/100 mL final
Tris-Cl
10 mM
0.1576 g
NaCl
10 mM
0.058 g
MgCl2(anhydrous)
3 mM
0.0287 g
Igepal CA-630
0.01%
10 uL
HCl
to pH 7.4
Wash buffer:
Component
Concentration
Amount/100 mL final
PBS
Base
SDS
0.01%
.01 g
EDTA
50 mM
1.461 g
2x TD buffer (from this nature paper):
Component
Concentration
Amount/L final
Tris
20 mM
3.264 g
MgCl2
10 m
0.95 g
DI H2O
main solvent
Acetic acid
to pH 7.6, before DMF addition
Dimethylformamide(DMF)
20% (v/v)
After cell fixation, permeabilize cells with lysis buffer for 10 minutes at room temperature.
Wash with PBS twice.
Prepare transpose mixture:
Component
Volume fraction
2x TD buffer
0.5
5 mM assembled Tn5
0.02
DI H2O
0.48
Add 50 uL of transpose mixture to cells to be transposed. Place the cells in a humid 37C box for 30 minutes.
For plated cells, wash with wash buffer three times, for 15 minutes each at 55C. For suspended cells, wash twice.
Add PBS media to cells and image/flow the resulting cells.