Isolation of Plant Nuclei Compatible with Microfluidic Single-nucleus ATAC-sequencing

Sandra B Thibivilliers; Dirk K Anderson; Marc Y Libault

doi:10.21769/BioProtoc.4240

Isolation of Plant Nuclei Compatible with Microfluidic Single-nucleus ATAC-sequencing

Bio Protoc. 2021 Dec 5;11(23):e4240. doi: 10.21769/BioProtoc.4240.

Authors

Sandra B Thibivilliers¹, Dirk K Anderson², Marc Y Libault¹

Affiliations

¹ Department of Agronomy and Horticulture, Centre for Plant Science Innovation, Center for Root and Rhizobiome Innovation, University of Nebraska-Lincoln, Lincoln, NE, USA.
² Flow Cytometry Service Center, Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.

Abstract

Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type. Recently, ATAC-seq experiments were conducted at the level of individual plant nuclei. Applying single nuclei ATAC-seq (sNucATAC-seq) technology to thousands of individual cells revealed more finely tuned profiles of chromatin accessibility. In this manuscript, we describe a method to isolate nuclei fom plant roots and green tissues, permeabilize the nuclear membrane using detergent to allow the penetration of the Tn5 transposase, and re-suspend them in a nuclei resuspension buffer compatible with the construction of sNucATAC-seq libraries using the 10× Genomic's Chromium technology. This protocol was successfully applied on Arabidopsis thaliana and Glycine max root nuclei.

Keywords: Chromatin accessibility; Fluorescence-activated nuclei sorting (FANS); Nuclei isolation; Plant single cell; sNucATAC-seq.