CUT&Tag of H3K27ac
Library strategy is CUT&Tag;1 million mESCs were grown for 24 hours and then induced with doxycycline (4 g/mL) for 24 hours. The cells were washed with PBS once and were sorted out by flow cytometry, and one hundred thousand MERVL-positive and MERVL-negative cells were collected for CUT&Tag (Novoprotein, N259-YH01). ConA-streptavidin beads were paired with living cells, and then incubated 2 hours in binding buffer with antibodies of interest 1:100 (H3K27ac Activemotif 39034, H3K4me1 Cell Signaling Technology 5326) at room temperature. Goat anti-rabbit IgG H&L 1:200 (Novoprotein, N269) was incubated for 1 hour at room temperature. Then pA-Tn5 was incubated for 1 hour at room temperature and used at 1:100. After washing steps and release from the beads, PCR was performed for 17 cycles. Samples were purified and sent for sequencing. All libraries were sequenced on the Illumina NovaSeq 6000 platform in 150-bp, paired-end format. Paired-end, raw sequencing reads were subjected to quality control and adapter trimming using the Trim Galore software (no version specified). The high-quality reads were aligned to the UCSC mm10 build using the Bowtie2 aligner (version 2.4.5), utilizing the parameters --end-to-end, --very-sensitive, and --no-mixed to ensure stringent alignment criteria. Picard(version 2.25) is used to remove duplicates in bam files, and then samtools(version 1.9) is used to extract results with MAPQ values greater than 30 for high-quality comparison results. samtools was used to index the results. Deeptools(version 3.5.5) was used to obtain bw files and calculate the enrichment of H3K27ac.