Researchers at Scipio Bioscience report progress in sample preparation for scRNA-seq based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, the researchers present a clinically relevant application of RevGel-seq for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles.
Sample preparation workflow and data analysis pipeline
(A) RevGel-seq workflow steps for sample preparation to characterize scRNA-seq. Barcoded beads and cells are attached via a bifunctional chemical linker. These complexes are dispersed in the hydrogel in the liquid state and immobilized upon gelation. Following cell lysis, RNA molecules are captured on the barcodes. After reverse transcription, barcoded cDNAs are PCR-amplified. (B) Workflow of the end-to-end data processing pipeline integrated in the Cytonaut platform. The pre-processing phase inputs the raw sequencing data (FASTQ files) and outputs quality indicators and count matrices, followed by the post-processing phase that inputs the count matrices to perform 2D embedding, cell clustering and differential gene expression. The Cytonaut Rover module enables interactive data visualization.
Overall, these results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.