GSM8670070: SW480 cell,KI-RPB1-HiBiT,50000_cells,LgBiT-Tn5(Af-CUT&Tag); Homo sapiens; 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. Following cell or nucleus counting, permeabilization is performed without the addition of antibodies. The permeabilized samples are incubated with LgBiT-Tn5 or NbALFA-Tn5, and unbound proteins are subsequently removed. A transposition activation reaction is then conducted to fragment intracellular DNA, which is extracted for downstream library preparation. Indexed libraries are generated using universal i5 primers and unique barcoded i7 primers, with each sample assigned a distinct barcode. The libraries are amplified through 15 cycles of PCR, and the resulting DNA is purified for high-throughput sequencing.