GSM8670033: mouse,pre-hepatectomy,ATAC; Mus musculus; ATAC-seq
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. Harvest cells by centrifuging at 600×g for 5 minutes, discard the supernatant, and resuspend in PBS. Centrifuge a single-cell suspension containing the desired number of target cells at 600×g for 5 minutes, discard the supernatant, and resuspend in 50 µL of pre-cooled lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.02% digitonin, 0.1% Tween-20). Allow the cells to lyse by placing them on ice for 5 minutes. Centrifuge at 500×g for 10 minutes at 4°C to collect the cell nuclei and immediately continue to transposition reaction. To make the transposition reaction mix, combine the following: 10 μL 5×TD Buffer (50 mM Tris pH 7.5, 25 mM MgCl2, and 50% dimethylformamide), 16.5 µL PBS, 0.5 µL 10% Tween-20, 0.5 µL 1% digitonin 18.5 ddH2O and 4 µL LgBiT/NbALFA-Tn5 Transposase (~1 µM). Resuspend nuclei in the transposition reaction mix and incubate the transposition reaction at 37°C for 30 min. PCR mix (Vazyme#TD601) and adapters (VazymeTD#202) were used for library amplification, the total volume is 50 µL. The fragmented DNA library was initially incubated with a 0.55× (27.5 µL) volume of DNA purification magnetic beads (Vazyme#N411). After discarding the magnetic beads, the supernatant was retained, and a 1.2× (60 µL) volume of DNA magnetic beads was added to further purify the DNA fragment library.