CUT & Tag of H3K18la in Y79（bw files）
Description
CUT&Tag assay was completed as previous described. 1×105 cells were harvested and washed using wash buffer (1×protease inhibitor cocktail, 20 mM HEPES, 0.5 mM spermidine and 150 mM NaCl). Activated concanavalin A-coated magnetic beads (Bangs Laboratories, Inc., Fishers, IN, USA) were added and incubated at room temperature for 20min. The mix was resuspended with dig wash buffer consisting of 2-mM EDTA. The cells were incubated with an anti-H3K18la and IgG antibody (12-370, Millipore) overnight at 4°C. The secondary antibody was added to the cells and incubated at room temperature for about 1h, 100 μl of pA-Tn5 adapter complex (40 nM) was subsequently added and incubated at room temperature for 1h. After washing, Tagmentation buffer was added and incubated at 37°C for 1h. Proteinase K was added and incubate at 55°C for 15min, then at 70°C for 20min. DNA was then extracted with AMPure XP beads (Beckham Counter) and eluted. For PCR, PCR was performed with the following thermocycler programme: 72°C for 5 min, 98°C for 30 s, 14 cycles of 98°C for 10 s and 63°C for 30 s, 72°C for 1 min, and finally holding at 8°C. The size distribution of libraries was assessed using Agilent 4200 TapeStation analysis, and libraries were combined to ensure uniform representation, targeting a final concentration as recommended by the manufacturer. Paired-end Illumina sequencing was conducted following the manufacturer’s guidelines. Alignment of paired-end reads was performed using Bowtie2 version 2.2. Peak calling was conducted using the following parameters: macs2 callpeak - t input_file - p 1e-5 - f BEDPE/BED (for Paired End vs. Single End sequencing data) - keep-dup all - n out_name. The criteria of fold change >2.0 (log2 ratio value > 1) and P value < 0.05 were employed to identify differentially accessible peaks.