A brand new spatial transcriptomics know-how—the DNA nanotechnology-driven technique referred to as “Gentle-Seq”—permits researchers to “geotag” the total repertoire of RNA sequences with distinctive DNA barcodes unique to a couple cells of curiosity. These goal cells are chosen utilizing mild beneath a microscope by way of a photocrosslinking course of.
With the assistance of DNA nanotechnology, the barcoded RNA sequences are translated into coherent DNA strands, which might then be collected from the tissue pattern and recognized utilizing next-generation sequencing (NGS). The Gentle-Seq course of may be repeated with totally different barcodes for various cell populations inside the identical pattern, which is left intact for follow-up evaluation.
This work is printed in Nature Strategies, within the paper, “Gentle-Seq: Gentle-directed in situ barcoding of biomolecules in fastened cells and tissues for spatially listed sequencing.”
“Gentle-Seq’s distinctive mixture of options fills an unmet want: the flexibility to carry out imaging-informed, spatially prescribed, deep-sequencing evaluation of arduous, if not impossible-to-isolate cell populations or uncommon cell sorts in preserved tissues, with one-to-one correspondence of their extremely refined gene expression state with spatial, morphological, and doubtlessly disease-relevant options,” stated Peng Yin, PhD, a core school member on the Wyss Institute.
Beforehand, Yin’s crew had developed the spatial transcriptomics technique SABER-FISH for imaging gene expression straight in intact tissues. “With SABER-FISH, we nonetheless had been orders of magnitude away from capturing cells’ full gene expression packages, with many 1000’s of various RNA molecules per cell. RNA molecules are simply too densely packed to be captured of their entirety utilizing current imaging strategies,” famous Jocelyn Kishi, PhD, a Wyss know-how growth fellow within the Yin lab. “Gentle-Seq solves this drawback by combining high-resolution barcode labeling with full-transcriptome sequencing through NGS, giving us the very best of each worlds and extra key benefits.”
“To particularly sequence the cells in custom-selected areas of intact tissue samples, we developed a brand new strategy for photocrosslinking DNA barcodes to copies of RNA molecules, and a DNA nanotechnology-powered process that makes them and their hooked up RNA sequences readable by NGS,” stated Ninning Liu, PhD, a postdoctoral fellow in Yin’s group.
First, DNA primers “base-pair” with RNA molecules in cells, and are prolonged to create cDNAs. Then, DNA barcode strands containing an ultrafast photocrosslinker nucleotide are base-paired to the cDNAs within the cells. These develop into completely linked collectively when a goal cell is lit up beneath the microscope by way of a stencil-like optical machine that retains different, nontarget cells within the microscopic area at nighttime and thus spares them from the photocrosslinking response. After washing the barcoded DNA sequences out of cells that weren’t completely linked in situ, the process may be repeated with totally different barcodes and light-weight patterns to label extra areas of curiosity.
“To have the ability to combine this barcoding workflow with NGS, we engineered a brand new stitching response that’s primarily based on DNA nanotechnology. This innovation permits us to transform our barcoded cDNAs into contiguous readout sequences. We will then extract the whole assortment of barcode-bearing cDNA sequences from the pattern, and analyze them with customary NGS strategies,” defined Sinem Saka, PhD, presently a gaggle chief on the European Molecular Biology Laboratory in Heidelberg, Germany and a former post-doc in Ying’s lab on the Wyss Institute. “Finally, every barcode traces the total transcriptome readout again to the pre-selected cells within the tissue pattern, which stays intact for subsequent analyses. This supplies us the distinctive probability to revisit the very same cells after sequencing for validation or additional exploration.”
Yin’s crew utilized Gentle-Seq to cross-sections of the mouse retina to profile three main layers with totally different features. The researchers reached a sequence protection akin to single-cell sequencing strategies and located that 1000’s of RNAs had been enriched between the retina’s three main layers. In addition they confirmed that after sequence extraction, the tissue samples remained intact and may very well be additional imaged for proteins and different biomolecules. Lastly, the crew was capable of isolate the total transcriptome of a dopaminergic amacrine cell of the retina—a uncommon cell sort that’s difficult to isolate.
“Our sequencing knowledge clearly confirmed that Gentle-Seq can decide pure variations within the construction of RNAs. Going ahead, we’re very concerned about utilizing Gentle-Seq to raised perceive the interaction between the immune system, disease-propagating cells, and totally different therapeutic methods akin to gene and cell remedy,” stated Kishi, who’s pursuing a path towards commercializing Gentle-Seq along with a few of her co-authors.