Light-Seq enabled the isolation of the full transcriptome of a very rare type of cell, known as “dopaminergic amacrine cell” (DAC, magenta), by retrieving merely four to eight individually barcoded cells per cross-section. DACs are extremely hard-to-isolate, also because of their intricate connections to other cells in the retina that were differently barcoded using Light-Seq. Credit: Wyss Institute at Harvard University
Viewing biological tissue under a microscope can allow researchers to observe the unique properties of different cell types, such as specific positions, unusual shapes or the presence of notable biomarkers. Utilizing sequencing methods to study the transcriptomes of specific cells or cell populations allows deeper insights into these cell types, but few methods allow for the preservation of spatial and morphological information of sequenced cells, and those that do have downsides such as destruction of the sample, high cost or limited depth and quality of analysis. Researchers from Harvard University’s Wyss Institute for Biologically Inspired Engineering have now developed a method that uses light to facilitate spatially-targeted sequencing of RNA in specific cells or cell populations, which exhibits similar performance to single-cell sequencing methods and keeps the sample intact for further analysis.
The technique, called Light-Seq, relies on the use of a photocrosslinking reaction to “geotag” RNA sequences with DNA barcodes under a microscope. First, reverse transcription is used to synthesize complementary DNA (cDNA) from the RNA sequences, then, a digital micromirror device (DMD) is used like an optical “stencil” to direct light in a specific area to facilitate the photocrosslinking reaction. Only cDNA sequences in the illuminated area are barcoded, allowing them to be spatially indexed in context of the imaged sample. Following this reaction, the unlinked barcodes can be washed away, and multiple rounds of light-directed barcoding can be carried out to target and index different cell populations. Lastly, a cross-junction synthesis reaction developed by the team allows the barcoded cDNA sequences and their barcodes to be copied into new single-stranded DNA that can be collected, amplified and sequenced using next-generation sequencing (NGS) techniques. This last step allows the sequences to be extracted without destruction of the sample.
The team first tested the technique using a mixed cell culture and were able to target, barcode and discriminate between mouse and human cells. They then applied Light-Seq to tissue cross-sections of mouse retina in order to profile three major cellular layers with different functions. The technique provided a sequence coverage comparable to single-cell sequencing methods, and the researchers were also able to isolate the full transcriptome of dopaminergic amacrine cells (DACs), a very rare cell type that is typically extremely difficult to isolate due to its intricate connections to other cells in the retina, explained study co-author Emma West. The team achieved this by targeting just four to eight individually barcoded cells per cross-section, said West. This study was published in Nature Methods.
“Light-Seq also picked up RNAs that were specifically expressed in DACs at low levels, as well as dozens of DAC-specific biomarker RNAs that, to our knowledge, had not been described before, which opens new opportunities to study this rare cell type,” noted West.
“Light-Seq’s unique combination of features fills an unmet need: the ability to perform imaging-informed, spatially prescribed, deep-sequencing analysis of hard, if not impossible-to-isolate cell populations or rare cell types in preserved tissues, with one-to-one correspondence of their highly refined gene expression state with spatial, morphological, and potentially disease-relevant features,” added Peng Yin, one of four corresponding authors on the paper.