GSM8670028: mouse,mYAP1-HiBiT,post-hepatectomy,LgBiT-Tn5(scAf-CUT&Tag); Mus musculus; OTHER
For cell samples, digestion followed by permeabilization is sufficient. For mouse liver tissue, 0.1-0.2 g of liver tissue was excised and washed with 2 mL of swelling buffer (10 mM Tric-HCl pH 7.5, 2 Mm MgCl2, 3 mM CaCl2, 1×Protease inhibitor cocktail). The liver was then dounced in 1 ml of swelling buffer (dounced 10 times, piston A) and incubated at 4°C for 20 min. Afterward, the liver was dounced again 20 times (piston B), and 1 mL of swelling buffer was added. The cells were filtered using a 100 μm cell strainer and spun at 400×g at 4°C for 10 min. The supernatant was removed, and the cells were resuspended in 0.5 ml of swelling buffer + 10% glycerol. Slowly vortex while adding drop by drop 0.5 mL of lysis buffer (Swelling buffer + 10% glycerol + 1% NP-40). Incubate for 5 min at 4°C and add 1.5 mL of lysis buffer. Spin at 600×g at 4°C for 5 min, remove the supernatant, and wash the nuclei again with 1.5 mL of lysis buffer. Spin at 600 g at 4°C for 5 min, remove the supernatant, and resuspend the nuclei in 1 mL of freezing buffer. Finally, the nuclei were stained with DAPI and counted. The LgBiT-Tn5 complex was prepared in 192-well PCR strip tubes, each containing 1.5 μL of 26 μM recombinant LgBiT-Tnp protein, 0.75 μL of 50 μM 5′ barcode, 0.75 μL of 50 μM 3′ barcode, and 4 μL of dialysis buffer, and incubated at 25°C for 60 minutes. HiBiT-tagged cells or nuclei (2 million) were washed, permeabilized, resuspended in antibody buffer with NanoLuciferase, and distributed into 192 wells (10,000 cells/nuclei per 50 μL). Each well received 1 μL of the LgBiT-Tnp complex, followed by incubation (room temperature for 60 minutes or overnight at 4°C). After centrifugation at 300×g for 3 minutes, the solution was mostly discarded, and the wells were washed three times with 100 μL Dig-300 buffer. The cells/nuclei were pooled, centrifuged at 600×g for 3 minutes, resuspended in 400 μL Dig-300 buffer, and filtered. Using FACS, 200 cells/nuclei were distributed into 96 wells, where tagmentation was initiated by adding 25 μL of tagmentation buffer, incubating at 37°C for 60 minutes, and stopping the reaction for DNA extraction. For library preparation, each well received 18 μL DNA, 1.25 μL of 10 μM 5′ library index barcode, 1.25 μL of 10 μM universal 5′ barcode, 0.5 μL TAE, and 5 μL of 5×TAB. PCR amplification was performed for 20 cycles.