Antibody-PA-Tnp Complex Formation (1 hour)
1. Mix the following in a microcentrifuge tube and incubate at room temp. for 10 min: 4.8 uL of H2O; 9 uL of 2X CB; 0.6 uL of 50 uM annealed 5' complex barcode; 0.6 uL of 50 uM annealed 3' complex barcode; 3 uL of 1 mg/mL recombinant PA-Tnp enzyme
2. Label a fresh 1.5 mL microcentrifuge tube for each antibody to be used. To each tube, add 1 uL of prepared PA-Tnp solution, 0.8 uL of 1X CB, and 0.8 uL of the desired antibody. (Multiply volumes by the number of replicates, if applicable.) Mix and incubate at room temperature for 30 min. Continue to the steps in the following section during this incubation time.
Cell Permeabilization (30 min to 1.5 hours)
Notes: two sets of instructions are provided for this section. One is for native ACT-seq and the other is for crosslinking ACT-seq (XACT-seq).
Unfixed permeabilized cells are fragile. For native ACT-seq, the centrifugation speed for your cell type needs to be determined before the protocol is run for the first time. It is recommended to permeabilize the cells in 1X CB, test various spin speeds, observe whether the cells pellet efficiently, and use the minimum effective speed for cell-spinning steps in the protocol after permeabilization. A swinging-bucket rotor is recommended to avoid cell loss during supernatant removal. 200 *g can be used as a starting point for optimization.
3. Harvest and transfer 1 million cells to a clean 1.5 mL tube. Wash cells with PBS to remove residual growth medium and/or trypsin solution. Pellet the cells.
4. Remove the supernatant and suspend the pellet in 1 mL of 1X CB. Incubate the tube on ice for 10 min to permeabilize the cells.
5. Spin down the cells (see the note on centrifugation above) and carefully remove the supernatant, leaving ~50 uL at the bottom of the tube to avoid cell loss.
6. Continue from step 12 below.
3. Harvest ~1 to 5 million cells in a 15 mL conical vial. Pellet the cells and aspirate the supernatant completely.
4. Suspend the pellet in 10 mL of room-temp. culture medium supplemented immediately prior to use with fresh 1% methanol-free formaldehyde. Rotate the tube for 10 min at room T.
5. Add 1/20 V of 2.5 M glycine. Rotate the tube for an additional 5 min at room T. Centrifuge at ~500 *g for 3 min at 4 °C.
6. Aspirate the supernatant and suspend the cells in 10 mL of cold PBS. Repeat centrifugation.
7. Repeat step 6. Suspend the cells in 1 mL of PBS and obtain a cell count.
8. Transfer 1 million cells to a clean 1.5 mL tube and centrifuge for 1 min at ~500 *g. (A swinging-bucket rotor is preferred for steps involving cell pellets in 1.5 mL tubes.)
9. Remove the supernatant and suspend the pellet in 1 mL of RIPA Buffer. Incubate the tube at room T for 10 min to lyse the cells and decondense the chromatin.
10. Spin down the cells at ~850 *g for 1 min. Carefully remove the supernatant, leaving ~50 uL at the bottom.
11. Suspend cells in 1 mL of 1X CB. Repeat the centrifugation and carefully remove the supernatant down to ~50 uL.
12. Suspend the cells in 1 mL of 1X CB. Aliquot 50 uL (~50 thousand cells) into new 1.5 mL tubes to accommodate the desired number of antibodies and replicates.
Complex Binding and Tagmentation (2.5 hours)
13. To each tube of cells, add 2.5 uL of the appropriate PA-Tnp-antibody complex and mix by pipetting gently. Incubate at room temperature for 60 min.
14. Add 500 uL of Wash Buffer to each tube and mix by inverting. Spin the cells and remove the supernatant as was done in step 5 (native ACT-seq) or step 8 (XACT-seq) above, leaving ~50 uL at the bottom to avoid loss of cells.
15. Add 500 uL of Wash Buffer. Rotate tubes for 5 min at room temperature. Spin and carefully remove supernatant as above.
16. Repeat step 15.
17. Dilute the samples to 100 uL with Wash Buffer and gently suspend the cells. Add 1 uL of 1 M MgCl2to each tube of cells and mix by pipetting. Incubate at 37°C for 60 min. Note: increasing this incubation time is not recommended as it may increase the background signal due to nonspecific Tn5 activity.
Library Preparation (2 hours)
Note: library index barcodes are different from the complex barcodes used above. See the Appendices document.
18. Stop the reaction by adding 4 uL of 0.5 M EDTA, 2 uL of 10% SDS and 1 uL of 20 mg/mL Proteinase K. Incubate at 55 °C for 60 min.
19. Purify the DNA using a Zymo ChIP Clean and Concentrator kit (or equivalent) and an elution volume of 10 uL.
20. To each sample, add 0.5 uL of library index barcode #1, 0.5 uL of library index barcode #2, and 10 uL of 2X NEB Phusion HF Master Mix (add last and mix). Amplify using the following program: 72 °C for 5 min; [98 °C for 10 s; 65 °C for 30 s; 72 °C for 15 s] (18 cycles for bracketed steps); and a final 72 °C for 5 min
21. Visualize the PCR products using gel electrophoresis. Excise gel fragments between about 200 to 800 bp and purify the DNA using a MinElute Gel Purification Kit (or equivalent). Libraries are ready for quantification and sequencing.