ATAC-seq Technique PDF

Technique - ATACseq ATAC-seq Assay for transposase- accessible chromatin with sequencing (next generation) Method to assess chromatin accessibility by mapping open chromatin regions using a transposase Applications of ATACseq Mapping chromatin accessibility Disease mec...

Technique - ATACseq ATAC-seq Assay for transposase- accessible chromatin with sequencing (next generation) Method to assess chromatin accessibility by mapping open chromatin regions using a transposase Applications of ATACseq Mapping chromatin accessibility Disease mechanisms, comparing different treatments in an experiment Identifying active regulatory elements (enhancers, promoters, etc) Cell-specific regulatory elements through single cell ATACseq Understanding transcription factor binding and footprints Learn about gene regulation networks Investigating chromatin modifications How chromatin modifications affect chromatin accessibility (in combination with ChIP-seq)  Open vs Closed Chromatin Mitotic chromosom Further e compaction 30nm fiber 10-11nm Closed fiber chromatin ATAC-seq allows detection of open areas of chromatin naked DNA 2nm diameter Open Naturally Occurring Transposons (transposable elements) Transposons: genetic elements that are able to move from one location in the genome to another location in the genome Transposable elements are abundant in plant and animal genomes, as well as in bacteria DNA transposons consist of: Transposase gene – encodes transposase enzyme which catalyzes all reactions necessary for DNA transposition Flanking regions of inverted terminal repeats which transposase binds to Additional sequences that are unrelated to transposition (ex.  Natural “Cut-and-paste” Transposition Transposase TIR = excises terminal Transposas transposon (cuts e inserts inverted within TIRs) repeats transposon into new Tnp = target site transposase gene is transcribed to enzyme (TNP) Original site of Transposase transposon recognizes and binds (double- to the terminal stranded break) inverted repeats is repaired Sinzelle et al., 200 Tn5 transposase used in ATACseq Derived from the Tn5 transposon in bacteria Commonly found in E. coli Formation of Tn5/Adapter Complex (Transposome) Next-generation adapter sequences are covalently attached to Tn5 transposase Adapters inserted into the DNA when the transposase acts Tn5/Adapter complex preferentially acts on open chromatin (nucleosome depleted) ATACseq Workflow Steps of ATACseq 1. Chromatin Preparation (cell lysis) Cells are collected and lysed to release chromatin Chromatin is kept intact to maintain natural nucleosome structure Steps of ATACseq 2. Transposase treatment Tn5/Adapter complex recognizes and cuts accessible (nucleosome free or sparse) regions of chromatin, creating double stranded breaks As chromatin is cut, Tn5 transposase simultaneously inserts sequencing adapters onto either side of the cut: tagmentation Steps of ATACseq 3. Purification and amplification DNA fragments with adapters are purified to remove any unwanted molecules (ex. untagged DNA, extra transposase) Purified DNA is amplified via PCR or bridge amplification, enriching the accessible chromatin regions 4. Next-generation sequencing Sequence is obtained for millions of short DNA fragments in parallel  Steps of ATACseq 5. Data Analysis Sequencing reads are mapped back to reference genome, identifying which areas of the genome are accessible and which are not Peaks represent regions of open/accessible chromatin (where more sequences were obtained) Compare differential accessibility between two experimental Steps of ATACseq 5. Data Analysis Small reductions in reads in an area of chromatin indicate a transcription factor footprint Binding of transcription factor decreases chromatin accessibility Motif analysis can be used on footprints to determine binding sequence and possibly identity of transcription factor Footprints can be validated with CHIP-seq Steps of ATACseq 5. Data analysis After determining identity of transcription factors, regulation cascades can be explored Learn more about genes, including the functions or Mouse tissue-specific chromatin accessibility Tissue-specific transcription factor enrichment based on motifs in accessible regions Limitations of ATACseq Limited resolution of nucleosome positions within open chromatin Limited information on chromatin structure or higher-order chromatin organization Single-cell ATACseq can have low sensitivity due to small amounts of chromatin Can detect chromatin availability due to nucleosomes, but can’t provide insight on other epigenetic marks such as DNA methylation Complex bioinformatics and difficulty interpreting regulatory function of accessible regions