Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies

Area of Science:

• Genomics and Molecular Biology
• Bioinformatics and Computational Biology

Background:

• Single-cell RNA sequencing (scRNA-seq) offers high-resolution phenotyping for complex biological samples.
• Widespread adoption of scRNA-seq necessitates standardized experimental design protocols.
• Challenges exist in optimizing sample preparation, sequencing, and data analysis for diverse research needs.

Purpose of the Study:

• To provide a comprehensive tutorial on single-cell RNA sequencing (scRNA-seq) experimental design.
• To guide researchers in making informed decisions regarding sample preparation, RNA sequencing, and data analysis.
• To facilitate the creation of broadly applicable scRNA-seq experimental frameworks tailored to specific research contexts.

Main Methods:

• Summarizes current methodological options for cell separation and single-cell capture at various resolutions and scales.
• Reviews RNA sequencing library preparation methods, highlighting features for informed selection.
• Provides a guide to major data processing steps and interpretation strategies for scRNA-seq data.

Main Results:

• Offers best practices for individual cell separation and an overview of capture technologies.
• Discusses critical features of RNA sequencing library preparation methods.
• Outlines essential data processing and interpretation guidelines to prevent erroneous analysis.

Conclusions:

• This tutorial serves as a reference for building robust single-cell experimental frameworks.
• It empowers researchers to tailor scRNA-seq experiments from sample preparation to data interpretation.
• Ensures meaningful data interpretation and avoids misinterpretations in complex biological studies.