A microfluidic platform towards automated multiplexed in situ sequencing

Abstract

Advancements in multiplexed in situ RNA profiling techniques have given unprecedented insight into spatial organization of tissues by enabling single-molecule quantification and sub-micron localization of dozens to thousands of RNA species simultaneously in cells and entire tissue sections. However, the lack of automation of the associated complex experimental procedures represents a potential hurdle towards their routine use in laboratories. Here, we demonstrate an approach towards automated generation and sequencing of barcoded mRNA amplicons in situ , directly in fixed cells. This is achieved through adaptation of a microfluidic tool compatible with standard microscope slides and cover glasses. The adapted tool combines a programmable reagent delivery system with temperature controller and flow cell to perform established in situ sequencing protocols, comprising hybridization and ligation of gene-specific padlock probes, rolling circle amplification of the probes yielding barcoded amplicons and identification of amplicons through barcode sequencing. By adapting assay parameters (e.g. enzyme concentration and temperature), we achieve a near-identical performance in identifying mouse beta-actin transcripts, in comparison with the conventional manual protocol. The technically adapted assay features i) higher detection efficiency, ii) shorter protocol time, iii) lower consumption of oligonucleotide reagents but slightly more enzyme. Such an automated microfluidic tissue processor for in situ sequencing studies would greatly enhance its research potentials especially for cancer diagnostics, thus paving way to rapid and effective therapies.

High-Resolution Transcriptomic and Proteomic Profiling of Heterogeneity of Brain-Derived Microglia in Multiple Sclerosis

“…In situ spatial detection methods are very suitable for regional validation of target genes in a cellspecific manner. ISS by CARTANA (Ke et al, 2013;Maïno et al, 2019) is a very promising and easy-to-use methodology, where individual genes are targeted by barcoded padlock probes, which is a more sensitive methodology compared to unbiased in situ approaches including FISSEQ (Lee et al, 2015). More specifically, the sensitivity of ST (7%), ISS (10%), and FISSEQ (0,005%) are all lower compared to single-cell or single-nuclei sequencing (20%).…”

Spatially Resolved Transcriptomes—Next Generation Tools for Tissue Exploration

“…[32] A couple of years later, in 2019, the ISS assay was automated on a microfluidic platform, significantly reducing hands-on time as well as the total protocol runtime. [33] A further development dubbed "HybISS" replaces SBL of RCPs with sequencing-by-hybridization (SBH). This method demonstrates significantly higher signal-to-noise ratio than the SBL counterpart and is thought to allow for larger gene panels.…”

Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue

“…Applying further advancements that have been incorporated, at least in part, to other spatial techniques to HybISS are currently being pursued to try to push the capabilities of the technology. This could be in the form of microfluidic devices to automate production ( 36 ), reducing time, or investigating how these cells are actually arranged in a three-dimensional space and not simply sequential sections.…”