Anti-H3K27ac, anti-H3K9/14ac, and anti-succinyllysine CutTag of Control, TGF-b1-treated or DLD-knocked down HGC27 cells
CUTTag genomic DNA extraction, library preparation and data processingControl, TGF-b1-treated or DLD-knocked down HGC27 cells (10,000) were prepared. Genomic DNA extraction and library preparation were performed with Hyperactive Universal CUTTag Assay Kit for Illumina (Vazyme). Cells were harvested at room temperature to avoid stress, and incubated with Concanavalin A beads, anti-H3K27ac, anti-H3K9/14ac, and anti-succinyllysine antibodies overnight at 4. Genomic DNA was fragmented by pA/pG-Tn5 transposon, and extracted using DNA Extract Beads. Spike-in DNA from E. coli was added to each sample for correction. DNA was used as a template for PCR amplification, and Illumina sequencing adapters were added using custom primers to generate the sequencing libraries. The library DNA was purified through DNA Clean Beads. Sequencing was performed by Illumina platform at a depth of 6 G per sample. The CUTTag fastq data were checked for quality control using fastqc software and was aligned to hg38 reference genome using Bowtie2. Spike-in calibration was performed for scaling by aligning whole sequence to target spike-in sequence. Format conversion was completed by bedtools. Scaled bigwig files were used to draw heatmaps using computeMatrix with region from 3000 bp around TSS. SERCA was used for peak calling in non-normalized and stringent model. Peaks were visualized using the IGV software.