Single‐cell RNA sequencing methodology, analysis, applications, and future directions with special focus on cotton

Single-cell RNA sequencing (scRNA-seq) has emerged as a revolutionary technology that has significantly increased our understanding of plant cellular diversity and gene expression. Unlike bulk RNA sequencing, scRNA-seq reveals gene expression profiles at the cellular level and identifies rare cell populations and complex regulatory networks. Innovations in single-cell isolation have addressed previous challenges unique to plant cells, such as large cell sizes and rigid walls. Data analysis pipelines have also improved quality control, normalization, clustering, and downstream analyses of high-dimension scRNA-seq data. These improvements enhance our understanding of plant morphogenesis and cellular heterogeneity, opening avenues for further investigation into the complex interplay between gene expression and plant development. This review explores recent advances in sample preparation, such as protoplast preparation and nuclei isolation, library preparation, sequencing, and a detailed data analysis pipeline. Further, we explored the diverse applications of scRNA-seq in the field of cotton research, such as fibre development, gland development, salt and stress responses, as well as elucidating molecular mechanisms in anther development and uncovering critical regulatory networks involved in plant regeneration. Despite the potential and recent advances of scRNA-seq, some challenges, such as protoplast preparation, cell size variability, and the requirement for reliable marker genes, still need to be addressed. Thus, future research should prioritize optimizing scRNA-seq methodologies, enhancing high-throughput capabilities, and integrating multi-omics approaches to address changing environmental conditions.