Identifying Differentially Expressed Genes Using Fluorescence-Activated Cell Sorting (FACS) and RNA Sequencing from Low Input Samples

Methods Mol Biol. 2018:1819:139-151. doi: 10.1007/978-1-4939-8618-7_6.

Abstract

Cell type-specific gene expression profiles are useful for understanding genes that are important for the development of different tissues and organs. Here, we describe how to perform fluorescence-activated cell sorting (FACS) on Arabidopsis root protoplasts to isolate specific cell types in the root. We then detail how to extract and process RNA from a very low number of cells (≥40 cells) for RNA sequencing (RNA seq). Finally, we describe how to process RNA seq data using TopHat and how to identify differentially expressed genes using PoissonSeq.

Keywords: Differential expression; Fluorescence activated cell sorting; Low input samples; Protoplasting; RNA sequencing.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Arabidopsis / cytology
  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Cytoplasm / metabolism*
  • Flow Cytometry / methods*
  • Gene Expression Regulation, Plant / physiology*
  • Plant Roots / cytology
  • Plant Roots / genetics
  • Plant Roots / metabolism*
  • RNA, Plant / biosynthesis*
  • RNA, Plant / genetics
  • Sequence Analysis, RNA / methods*

Substances

  • RNA, Plant