ATAC-seq

Technical Background

During the DNA replication or transcription in eukaryotic cells, trans-acting factors may bind to cis-acting elements in open chromatin regions, such as promoters, enhancers, insulators, silencers, etc., thereby regulating gene expression in the cell. This characteristic of chromatin is referred to as "Chromatin Accessibility" in biology. Accurately and quantitatively analyzing chromatin accessibility has become crucial for studying gene expression regulation and various biological processes.

Currently, the main technologies for studying chromatin accessibility include nucleic acid cleavage-based MNase-seq, DNase-seq, ATAC-seq, and phenol-chloroform extraction-based FAIRE-seq. Among these, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technology researched and developed in 2013 by the team of William Greenleaf and Howard Y Chang at Stanford University to study chromatin accessibility using the NGS library "Magic Cleavage" - Tn5 transposition enzyme. Compared with FAIRE-seq, MNase-seq, and DNase-seq, ATAC-seq has become the most efficient high-throughput technology for studying chromatin accessibility due to its advantages of simple experimental library building, shorter processing cycle, and minimal cell requirements.

ATAC-seq Technical Principle

First, isolate cell nuclei and incubate them with Tn5 enzyme carrying the labeled sequence. At this stage, the Tn5 enzyme may fragment the DNA in open chromatin regions and add the labeled sequence to the ends; Next, use primers designed based on the labeled sequence to perform PCR amplification; Finally, subject the amplified products to high-throughput sequencing.

ATAC-seq Technical Principle

Sequencing data quality control, reference genome alignment, and library quality evaluation, including inserted fragment length distribution, TSS enrichment score, library complexity (NRF, PBC1, PBC2),etc.;

Peak calling, including evaluation of reproducibility of biological replicate samples (PCA, correlation heat maps), and IDR analysis;

Identification and functional annotation of accessible regions of differential chromatin, and analysis of transcription factor binding motifs, and transcription factor footprints.

Integration of multi-omics analyses, including combining data from Whole Genome Sequencing (WGS), RNA-seq, ChIP-seq, CUT&Tag, and 3D genomics technologies (Hi-C and HiChIP) to analyze the genetic basis of phenotypic variation.

1. Suitable for various types of samples, any eukaryotic fresh tissue cells or even frozen tissue cells can be used;
2. High sensitivity, high signal-to-noise ratio;
3. The initial cell demand is low, the number of cells is generally >50.000, and in special cases it can be as low as 500 cells
4. Experimental library preparation is simple.

I. Application Scenarios

1. Generate chromatin accessibility maps and identify cell/tissue-specific regulatory elements and transcription factors;

2. Perform differential analysis of chromatin accessibility to reveal the epigenetic basis of phenotypic variation through transcriptional regulation;

3. Integrate multi-omics data and establish a transcriptional regulatory network to facilitate the discovery of key genetic variations of biological phenotypes.

II. Application Cases

1. Application of ATAC-seq in Agricultural Animal and Plant Research

Annotation of functional elements in agricultural animal genomes and the study of their spatial conformation regulation patterns are essential foundations for understanding the genetic mechanisms of important traits. In 2021, researchers employed a combination of omics technologies, including ATAC-seq, ChIP-seq, RNA-seq, and Hi-C, to study four pig breeds, namely big lean white pigs, Duroc pigs, fatty Enshi black pigs and Meishan pigs, and obtained 199 sets of high-quality epigenomic data from 12 different tissues. With in-depth analysis, over 220,000 genome regulatory elements and 3,316 new transcripts were identified and functional features of super-enhancers, active promoters, and other regulatory elements were also elucidated. Further integration with Genome-wide Association Analysis (GWAS) results showed the systematic description of the genomic distribution characteristics of SNPs associated with important pig traits in relation to regulatory elements. The study revealed significant enrichment of GWAS association signals near enhancers and identified causal mutations for some important trait candidates. This achievement has provided robust data support for breeding using whole-genome functional mutation loci.

2. Application of ATAC-seq in Regeneration Research

The regenerative capacity of callus is one of the core scientific questions in the field of animal and plant regeneration. In 2019, researchers took the hofstenia miamia as the study object and employed ATAC-sqe technology based on the reference genome assembled from scratch generate the dynamic map of chromatin accessibility during the regeneration process.

1. A high-quality reference genome forhofstenia miamia(950 Mb) has been assembled;

2. ATAC-seq data at different time points during tail regeneration has been analyzed and approximately 18,000 differential open chromatin regions have been identified (adjusted p < 0.05, Wald test);

3. Motif analysis of differential open chromatin regions revealed that the binding sites of early growth response (EGR) factors exhibited the most significant dynamic changes throughout the regeneration process. The EGR motif was in a closed state at 0 hours, opened at 3 hours, and remained open for 48 hours;

4. By inhibiting EGR expression through RNAi at 6h and combining it with RNA-seq, they discovered a series of potential EGR target genes. Further, by integrating ATAC-seq data, they focused on target genes with both EGR binding sites and reduced chromatin accessibility after RNAi, suggesting that EGR can directly bind to its own regulatory regions and those of other injury response genes to influence the regeneration process.