See Appendix A in the supplemental file for expression and purification of recombinant PA-Tnp.
See Appendix B in the supplemental file for instructions on preparing the annealed complex adapters.
Antibody-PA-Tnp Complex Formation (1 hour)
1. For each PA-Tn5 complex adapter pair, mix the following in a microcentrifuge tube and incubate at room temp. for 10 min:
4.5 uL of 2X CB + PI
2 uL of 50 uM annealed complex adapter A
2 uL of 50 uM annealed complex adapter B
2.5 uL of 1 ug/uL recombinant PA-Tn5 enzyme
2. Label a set of microcentrifuge tubes for each antibody/PA-Tn5 complex combination. To each of these tubes, add 1.5 uL of the matching complex from step 1, 1.5 uL of 2X CB + PI, and 1.5 uL of the desired antibody. (Multiply these volumes for the number of replicates, if applicable.) Mix by pipetting and incubate at room temperature for 30 min.
Cell Permeabilization (30 min)
See Appendix C in the supplemental file for instructions on preparing crosslinked cells. If using frozen crosslinked cells, thaw a cell pellet equivalent to 1 million cells on ice and proceed immediately to step 4 below.
If starting with fewer than ~1 million cells, scale the final volume used in step 7 appropriately.
3. Transfer 1 million cells to a clean 1.5 mL tube and centrifuge for 2 min at 500 *g.
4. Remove the supernatant and suspend the pellet in 1 mL of freshly prepared RIPA Buffer. Incubate the tube at room T for 10 min to lyse the cells and decondense the chromatin.
Note: a swinging-bucket rotor is strongly recommended for the remaining centrifugations!
5. Spin down the cells at 850 *g for 2 min. Carefully remove the supernatant, leaving ~50 uL at the bottom to avoid loss of cells.
6. Suspend the cells in 1 mL of Wash Buffer. Repeat the centrifugation and carefully remove the supernatant down to ~50 uL
7. Suspend the cells in 1 mL of Wash Buffer. If you started with fewer than ~1 million cells, make sure the final cell concentration is equivalent.
Complex Binding and Tagmentation (2 hours)
8. Label a 1.5 mL tube for each sample. Transfer a 50 uL cell aliquot (~50 thousand cells) into each tube. Add 4.5 uL of the matched AB-Tnp complexes from step 2 for the first binding step. Mix each sample by pipetting gently. Incubate the samples at room temperature for 30 min.
9. Add 1 mL of Wash Buffer to each tube. Rotate the tubes for 5 min at room temperature. Centrifuge the cells at 850 *g. Remove the supernatant, leaving ~50 uL at the bottom to avoid loss of cells.
10. Add 3 uL of 50 uM annealed blocking adapter to the sample and mix by pipetting. Incubate for 10 min at room temperature.
11. Add 4.5 uL of the next AB-Tnp complex to each sample and mix by pipetting gently. Incubate at room temperature for 30 min.
12. If probing more than two marks per sample, repeat steps 9 through 11 for each remaining complex.
13. Add 500 uL of Wash Buffer to each tube and rotate the tubes for 5 min at room temperature. Centrifuge the cells at 850 *g and remove the supernatant down to ~50 uL.
14. Repeat step 13.
15. Dilute the samples to ~100 uL with Wash Buffer using the volume markings on the sides of the tubes. Add 1.5 uL of 1 M MgCl2 to each tube of cells and gently suspend the cells by pipetting. Incubate the cells at 37°C for 60 min to allow targeted tagmentation to occur.
Note: extending this reaction time is not recommended as it may increase the background signal at accessible chromatin regions due to nonspecific transposase activity.
Sample Purification (1.5 hours)
16. Stop the reactions by adding 8 uL of 0.5 M EDTA. Vortex thoroughly to mix. Incubate samples at 80 °C for 5 min.
17. Add 2 uL of 10% SDS and 1 uL of 20 mg/mL Proteinase K. Incubate at 55 °C for 60 min.
18. Purify the DNA using a MinElute PCR Purification kit (or equivalent) and an elution volume of 20 uL.
19. Store the purified samples at -20°C or -80°C or proceed to the following section.
Library Preparation (1 hour)
Note: the library PCR adapters are not the same as the complex adapters used in step 1! Use barcoded Illumina NextEra PCR primers or equivalent.
20. Transfer each ~20 uL sample to a PCR tube. To each sample, add 0.5 uL of 50 uM PCR adapter #1, 0.5 uL of 50 uM PCR adapter #2, and 20 uL of 2X NEB Phusion HF Master Mix. Mix by pipetting and amplify using the following program:
72 °C for 5 min
98 °C for 10 s |
65 °C for 30 s | 15 to 17 cycles
72 °C for 15 s |
72 °C for 5 min
21. Visualize the PCR products using gel electrophoresis (e.g. on a 2% E-gel run for 20 min). Visible smears and bands above ~250 bp indicate a detectable signal.
22. Excise gel slices corresponding to DNA fragment sizes between ~250 to 800 bp. Purify the PCR products using a MinElute Gel Purification Kit (or equivalent). The DNA libraries are ready for quantification, multiplexing, and sequencing.