Spatiotemporal Developmental Trajectories in the Arabidopsis Root Revealed Using High-Throughput Single Cell RNA Sequencing

A single cell RNA sequencing atlas of the Arabidopsis root distinguishes cells both by developmental fate and time, revealing defining expression features that depict a complex cascade of developmental progressions from stem cell through differentiation supported by mirroring waves of transcription factor expression. Methods: mRNA profiles of 6-day-old wild-type (WT) and shortroot-knockout Arabidopsis thaliana roots were generated by deep sequencing of single cell (all) and bulk RNA libraries (wild type only), in duplicate (bulk & wild-type single cell) and singlicate (shr-3), using Illumina NextSeq. The sequence reads that passed quality filters were analyzed at the transcript level. Single Cell libraries were processed and analysed using Cell Ranger, STAR, Seurat. Bulk libraries were processed using Trimmomatic, STAR, HTSeq and DEseq2. Results: For Single Cell - Using an optimized data analysis workflow, we mapped ~87k sequence reads per wild-type cell to the Arabidopsis genome (TAIR10) and identified a median of 4,276 genes and 14,758 transcripts per cell. In total, transcripts for 16,975 genes were detected (RPM =1) from wild-type cells. After correction for read depth, this represents ~90% of genes detected by bulk RNA-seq of protoplasted root tissue. Bulk RNA-seq data identified genes induced by protplasting of the Arabidopsis root which could be discounted from single cell analysis. The global gene expression profiles of pooled scRNA-seq and bulk RNA-seq are highly correlated (r = 0.9) indicating that plant scRNA-seq is highly sensitive. Conclusions: Our high-resolution single cell RNA sequencing atlas of the Arabidopsis root captures precise temporal information for all major cell types, revealing new regulators and defining features foreach. Developmental trajectories derived from pseudotime analysis depict a finely resolved cascade of developmental progressions between stem cell and final differentiation supported by mirroring waves of transcription factor expression. Overall design: Single cell RNA sequencing for 5,767 cells (total) from Arabidopsis (2x wild-type root replicate, 1x shr-3 mutant root replicate). Bulk RNA sequencing for Arabidopsis 2x replicates unprotoplasted roots and 2x replicates protoplasted roots.

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Denyer T, Ma X, Klesen S, Scacchi E, Nieselt K, Timmermans MCP