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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...

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Updated: Jun 26, 2026

Transcriptome Analysis of Single Cells

Transcriptome Analysis of Single Cells

Published on: April 25, 2011

An Efficient and Universal Protoplast Isolation Protocol Suitable for Transient Gene Expression Analysis and

Juanjuan Wang^1,2, Yang Wang^1,2, Tianfeng Lü¹

¹State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.

International Journal of Molecular Sciences

|April 12, 2022

Summary

Researchers developed a new protocol to isolate plant protoplasts, enabling single-cell RNA sequencing (scRNA-seq) for studying plant development. This method works across diverse species and organs, overcoming previous limitations in plant cell isolation.

Keywords:

Chirita pumila cell walls protocol protoplast isolation single-cell RNA sequencing transient gene expression

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Area of Science:

Plant Biology
Molecular Biology
Genomics

Background:

Single-cell RNA sequencing (scRNA-seq) offers high-resolution insights into complex organ development by tracking cell differentiation.
Isolating intact plant cells (protoplasts) is challenging due to rigid cell walls, hindering scRNA-seq applications in many species.

Purpose of the Study:

To establish an efficient and universally applicable protocol for preparing viable plant protoplasts.
To enable advanced single-cell analyses, including scRNA-seq, in diverse plant species.

Main Methods:

A two-step enzymatic digestion protocol using different buffer compositions was developed for protoplast isolation.
The protocol was optimized for *Chirita pumila* and validated on multiple organs from various angiosperm species.

Main Results:

Successful isolation of viable protoplast suspensions suitable for downstream expression analyses.
Demonstrated broad applicability across different plant organs (petals, fruits, roots, gynophores) and species.
High-quality protoplasts were obtained, facilitating scRNA-seq.

Conclusions:

This robust protocol overcomes significant barriers in plant protoplast isolation.
It opens new avenues for studying cell-type specification, tissue function, and organ diversification in plants using single-cell technologies.