Fig. 1

Schematic overview of single-cell ATAC-seq assays and analysis steps. a Single-cell ATAC libraries are created from single cells that have been exposed to the Tn5 transposase using one of the following three protocols: (1) Single cells are individually barcoded by a split-and-pool approach where unique barcodes added at each step can be used to identify reads originating from each cell, (2) microfluidic droplet-based technologies provided by 10X Genomics and BioRad are used to extract and label DNA from each cell, or (3) each single cell is deposited into a multi-well plate or array from ICELL8 or Fluidigm C1 for library preparation. b After sequencing, the raw reads obtained in .fastq format for each single cell are mapped to a reference genome, producing aligned reads in .bam format. Finally, peak calling and read counting return the genomic position and the read count files in. bed and .txt format, respectively. Data in these file formats is then used for downstream analysis. c ATAC-seq peaks in bulk samples can generally be recapitulated in aggregated single-cell samples, but not every single cell has a fragment at every peak. A feature matrix can be constructed from single cells (e.g., by counting the number of reads at each peak for every cell). d Following the construction of the feature matrix, common downstream analyses including visualization, clustering, trajectory inference, determination of differential accessibility, and the prediction of cis-regulatory networks can be performed using the methods benchmarked in this manuscript