GSM6040545: WT_TLE3 CUTnTag; Mus musculus; OTHER
GSM6040545
Siglec-F(-)Ly6C(lo)CD64(+) colonic macrophages were sorted from male WT (ten mice) and pooled together. Cells were divided into two aliquots, 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-IgG (CST 66362) or anti-TLE3 (Proteintech 11372-1-AP) 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 (20 cycles); and 72°C 5 min. Finally, the libraries were purified using DNA clean beads (Vazyme).