ATAC‑seq (Assay for Transposase‑Accessible Chromatin) is a high‑throughput sequencing technique based on Tn5 transposase that detects chromatin open regions genome‑wide, thereby revealing transcriptional regulatory activity. This course systematically covers its core principles: live cells are fixed and gently permeabilized to allow Tn5 transposase to enter the nucleus and specifically cleave nucleosome‑depleted regions; during cleavage, sequencing adapters are directly added to both ends of the fragmented DNA. After PCR amplification and library construction, high‑throughput sequencing is performed. The data analysis pipeline includes quality control of raw reads, alignment to the reference genome (MapQ ≥ 30), removal of PCR duplicates, and peak calling using MACS2 (with FDR control). Differential open peaks between samples are identified using DiffBind and DESeq2 (|log₂FC| ≥ 1, FDR ≤ 0.01). Genes associated with differential peaks are subjected to GO and KEGG enrichment analyses (Fisher’s exact test), and motif discovery is carried out with HOMER for both de novo prediction and known database matching to infer potential transcription factor binding. The course also compares ATAC‑seq with CUT&Tag (targeting specific proteins), DNase‑seq, MNase‑seq, and FAIRE‑seq, highlighting ATAC‑seq’s advantages in requiring fewer cells, shorter experimental time, high sensitivity, and good reproducibility, especially suitable for screening regulatory elements and transcription factor binding sites in the absence of specific antibodies. Combining theory with case studies, this course provides systematic methodological guidance for researchers in epigenetics and gene regulation.