RNA sequencing in situ

Writing in Science, Lee et al. report an exciting first step toward combining some of the best aspects of both methods to sequence RNA in single cells in situ. Their approach, called fluorescence in situ RNA sequencing (FISSEQ), effectively treats the cell as a sequencing chip while preserving spatial information about RNA location.

The authors begin by fixing cells, reverse transcribing the RNA in situ and copying the resulting cDNAs by rolling-circle amplification to generate single-stranded DNA ‘nanoballs’ . Each nanoball, about 200–400 nm in diameter, consists of many cDNA copies of an original RNA sequence. The large number of repeats means that the nanoballs can withstand repeated sequencing reactions, allowing the authors to analyze them inside cells using sequencing by oligonucleotide ligation and detection (SOLiD). In this sequencing method, fluorescent probes are sequentially hybridized to the nanoballs, with a color corresponding to a pair of adjacent bases, and the sequence of colors is used to determine the sequence of the transcript. Because this process involves repeated imaging of the sample, careful image analysis is needed to reconstruct the sequences.