Profiling single-cell chromatin accessibility in plants

Abstract

Coupling assay for transposase-accessible chromatin sequencing (ATAC-seq) with microfluidic separation and cellular barcoding has emerged as a powerful approach to investigate chromatin accessibility of individual cells. Here, we define a protocol for constructing single-cell ATAC-seq libraries from maize seedling nuclei and the preliminary computational steps for assessing data quality. This protocol can be readily adapted to other plant species or tissues with minor changes to reveal chromatin accessibility variation among individual cells. For complete details on the use and execution of this protocol, please refer to Marand et al. (2021).

Keywords: developmental biology; genomics; molecular biology; plant sciences; sequence analysis; sequencing; single cell.

Graphical abstract

Figure 1

Sample preparation (A) Seven-day old B73 maize leaf roll on pitri dish. (B) Samples are saturated with 500 μL nuclear lysis buffer. (C) Homogenized leaf tissue following two minutes of chopping in nuclear lysis buffer. (D) Filtered lysate containing DAPI, nuclei, chloroplast, and mitochondrial contamination prior to flow sorting.

Figure 2

Flow sorting gating strategy (A) Forward scatter versus side scatter to eliminate debris. (B) DAPI width versus DAPI area to remove nuclear aggregates. (C) Green fluorescence versus DAPI area to exclude green fluorescing material. (D) Distribution of events by DAPI area to collect nuclei at various stages of the cell cycle and ploidy.

Figure 3

Evaluating DAPI-stained nuclei quality under a fluorescent microscope (A) 20× magnification of DAPI-stained nuclei via epifluorescence (top) and bright field (bottom) microscopy. The scale bar indicates 20 μm. (B) Intact nuclei at 40× magnification via epifluorescence (top) and bright field (bottom) microscopy. (C) Nuclei with intermediate ruptured nuclear envelopes at 40× magnification via epifluorescence (top) and bright field (bottom) microscopy. (D) Lysed nuclei at 40× magnification via epifluorescence (top) and bright field (bottom) microscopy.

Figure 4

Fragment distributions of scATAC-seq libraries (A) High quality scATAC-seq library. LM; Lower marker (1 bp). UM; Upper marker (6,000 bp). (B) Poor quality scATAC-seq library. RFU; relative fluorescence units.

Figure 5

Quality control and initial scATAC-seq data processing (A) Identification of high-quality barcodes using spline-fitting and signal enrichment at TSSs and ACRs. FRiPs; Fraction reads in peaks. (B) Sparse matrix filtering to remove low frequency features (1-kb bins) and nuclei with few accessible features. (C) Visualization of nuclei similarity colored by graph-based clusters (Louvain) of genome-wide chromatin accessibility profiles.