BenchMasteryMolecular biology techniques & virtual lab

ATAC-seq

Tagmentation of tens of thousands of nuclei in one tube, giving one averaged accessibility profile per sample. The Omni-ATAC variant adds digitonin and Tween-20 and a wash step, which lowers mitochondrial reads and background and works on frozen tissue.

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Bulk ATAC

Tagmentation of tens of thousands of nuclei in one tube, giving one averaged accessibility profile per sample. The Omni-ATAC variant adds digitonin and Tween-20 and a wash step, which lowers mitochondrial reads and background and works on frozen tissue.

How it differs from the standard method

  • About 50,000 nuclei per standard reaction; scale the Tn5 if you have far fewer.
  • Quality judged by the fragment ladder, TSS enrichment, FRiP and mitochondrial fraction.
  • Paired-end sequencing gives fragment sizes, which separate nucleosome-free and nucleosomal signal.
In this lesson · about 2 min read
  1. Let a transposase find the open chromatin
  2. The ratio of nuclei to Tn5
  3. Judging a library
  4. Single-cell ATAC
  5. Key terms
  6. When to use it
  7. Sub-techniques

1Let a transposase find the open chromatin

ATAC-seq (Assay for Transposase-Accessible Chromatin) uses a hyperactive Tn5 transposase loaded with sequencing adapters. Added to intact nuclei, it cuts DNA and pastes an adapter into the cut in one step, but it can only reach DNA that is not wrapped around a nucleosome or covered by a bound protein.

< 100 bp

Nucleosome-free fragments

Two cuts within one open stretch of DNA. They carry most of the signal at promoters and enhancers and are what peaks are mostly made of.

~200, 400, 600 bp

Nucleosomal ladder

Cuts in the linkers on either side of one, two or three nucleosomes. A clear ladder is the sign of intact chromatin and a balanced amount of Tn5.

TSS

Promoter enrichment

Insertions pile up just upstream of active transcription start sites. Their aggregate height over the ±2 kb flanks is the TSS enrichment score.

2The ratio of nuclei to Tn5

The reaction is sized for a number of nuclei. With too few nuclei, the excess enzyme cuts into nucleosomal DNA as well: fragments get shorter, the ladder smears out, and the signal at open sites is diluted by cuts everywhere.

3Judging a library

  • A nucleosome ladder on the fragment-size trace and in the sequenced insert sizes.
  • TSS enrichment: about 7 or more is good on a GRCh38 RefSeq-style annotation; values under ~5 mean high background. The exact cut-offs depend on the annotation used.
  • Fraction of reads in peaks above about 20%, ideally 30% or more.
  • Few mitochondrial reads, and enough non-duplicate nuclear fragments (around 25 million or more per replicate).
  • Peaks at known promoters and enhancers, and reproducibility between replicates.

4Single-cell ATAC

Nuclei are tagmented in bulk and then partitioned into droplets (or split-and-pooled through wells) so that every fragment carries a barcode from its nucleus. Each nucleus has only two copies of each site, so one cell's profile is sparse: thousands to tens of thousands of fragments, most sites seen once or not at all.

Key terms

Practise these as flashcards →
Tn5 transposase

An enzyme that cuts DNA and inserts the adapters it carries in one step (tagmentation); in ATAC it only reaches accessible DNA.

Nucleosome-free region (NFR)

Open DNA between nucleosomes, typically at promoters and enhancers; gives fragments shorter than about 100 bp.

TSS enrichment

Insertions at transcription start sites relative to the flanks 2 kb away; a measure of signal over background.

FRiP

Fraction of reads in called peaks.

Mitochondrial fraction

Share of reads from mitochondrial DNA, which is discarded; high values waste sequencing.

Doublet

Two nuclei that share one barcode in a single-cell experiment; their profile is a mixture.

Footprint

A small dip in insertions where a bound transcription factor protects its motif from Tn5.

When to use it

  • To map accessible chromatin genome-wide from small numbers of cells, quickly and without an antibody.
  • To compare regulatory landscapes between conditions or cell types and pick candidate enhancers and transcription factors.
  • Single-cell ATAC when the population is mixed and the regulatory programmes of individual cell types are the question.

Compared with alternatives

Protein–DNA binding maps →

Maps one protein or histone mark with an antibody; use it once ATAC suggests which factor matters.

Methylation sequencing →

Reads DNA methylation, a different and more stable layer of regulation.

RNA-seq →

Measures the transcripts that open chromatin may or may not lead to; often paired with ATAC.

Single-cell RNA-seq →

Cell types by expression rather than accessibility; multiome kits read both from the same nucleus.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Tagmentation of tens of thousands of nuclei in one tube, giving one averaged accessibility profile per sample. The Omni-ATAC variant adds digitonin and Tween-20 and a wash step, which lowers mitochondrial reads and background and works on frozen tissue.

  • About 50,000 nuclei per standard reaction; scale the Tn5 if you have far fewer.
  • Quality judged by the fragment ladder, TSS enrichment, FRiP and mitochondrial fraction.
  • Paired-end sequencing gives fragment sizes, which separate nucleosome-free and nucleosomal signal.