Antibody-guided Chromatin Tagmentation for Two or More Factors (ACT2-seq)


 This protocol details the reagents and steps required to perform antibody-guided chromatin tagmentation for two or more factors (ACT2-seq, ACT2). Like its predecessor ACT-seq, ACT2 uses a fusion of protein A and Tn5 transposase to bind and profile epigenetic marks across the genome. ACT2 builds on the capabilities of ACT-seq by directly and concurrently profiling co-occupancy of epigenetic marks, which previously required laborious, expensive, and technically challenging approaches involving fluorescence, magnetic beads, or single-cell methods. ACT2 requires only standard pipetting and centrifugation techniques and can be completed in less than a single day of bench work.

Add protease inhibitors to a 500 mL aliquot immediately before use. See Appendix C in the supplemental le 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 nal 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 nal 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 rst 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.
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 MgCl 2 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 nonspeci c transposase activity.
Sample Puri cation (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.
18. Purify the DNA using a MinElute PCR Puri cation kit (or equivalent) and an elution volume of 20 uL.
19. Store the puri ed samples at -20°C or -80°C or proceed to the following section. This indicates that the protocol did not work correctly, but does not give any information as to what step was responsible. If you encounter this issue, we recommend running a positive-control sample generated using either H3K4me3 or H3K27ac alongside your experimental samples. If there is no visible signal in the positive control lane, it indicates an issue with the cells or experimental steps. If you aren't already, de nitely consider trying a swinging-bucket rotor to maintain cell number throughout the experiment.