GSM6040543: WT_H3K27ac CUTnTag; Mus musculus; OTHER
GSM6040543
Siglec-F(-)Ly6C(lo)CD64(+) colonic macrophages were sorted from male WT (four mice) and Tle4cKO (four mice) mice at the same time. Cells from mice of same genotype were pooled together. Cells were washed once with Wash Buffer and incubated with ConA-coated magnetic beads for 10 minutes at room temperature (RT). After magnetic separation, ConA bead-bound cells were resuspended in Antibody Buffer containing anti-H3K27Ac (Abcam ab4729) primary antibody and incubated overnight at 4°C. The solution was removed by magnetic separation, and an anti-rabbit secondary antibody (Vazyme, Nanjing, China) in Dig-Wash Buffer were added to each reaction, which was followed by 1-hour incubation at RT. Unbound antibodies were removed by washing with Dig-Wash Buffer, and cells were then incubated with pA-Tn5 for 1 hour at RT. The tagmentation reaction was activated by MgCl2 and terminated by the addition of EDTA, SDS, and Proteinase K. DNA fragments were purified by phenol-chloroform method and amplified by PCR (Vazyme) using the following PCR conditions: 72°C for 3 min; 98°C for 30 s; thermocycling at 98°C for 15 s, 60°C for 30 s and 72°C for 30 s (15 cycles); and 72°C 5 min. Finally, the libraries were purified using DNA clean beads (Vazyme).