Single-protoplast and single-nucleus sequencing of the leaves of two C4 species: Gynandropsis gynandra and Flaveria bidentis

In this study, we optimized protocols for single-nucleus and single-protoplast RNA sequencing (RNA-seq) to generate high-resolution transcriptome atlases for two C4 plant species, Gynandropsis gynandra and Flaveria bidentis. Using Chromium (10X Genomics) technology, we generated single-nucleus and single-protoplast RNA-seq libraries for both species and mapped raw reads to their respective genomes. After quality control, doublet removal, and batch correction, we produced gene expression matrices and performed clustering analysis. We identified major leaf cell types, confirmed the known patterns of C4 gene compartmentalization, and identified conserved and species-specific transcription factors that may contribute to C4 evolution. Our dataset provides a valuable resource for studying the regulatory dynamics of C4 photosynthesis at cellular resolution and offers insights into the evolution of C4 traits.