PROBE-BASED DEVICE-FREE SINGLE-CELL RNA PROFILING

PROBE-BASED DEVICE-FREE SINGLE-CELL RNA PROFILING

Researchers at UCSF have developed a novel method for probe-based scRNA-seq.

Single cell RNA sequencing (scRNA-seq) has revolutionized the field of cell biology by enabling nucleic acid profiling at single cell resolution. Typical detection efficiency of scRNA-seq ranges between 5-45%, mostly due to template switching inefficiency during reverse transcription. This limitation proves particularly problematic for detecting low copy number RNAs, characterizing cell populations with few cells, and subtle shifts in phenotype. In another vein, single molecule fluorescence in situ hybridization (smFISH) regularly achieves detection efficiency of nearly 100% due to the utilization of multiple probes that directly target RNA. This method can be performed on a single cell level with multiple probes for a given transcript, such as in the methods HyPR-Seq and ProBac-seq. However, these methods have inherent limitations including their reliance on hybridization-based specificity, which in turn leads to low specificity. Additionally, their use of microfluidic partitioning of cells limits the number of cells that can be profiled in a single experiment and requires the addition of costly and complex instrumentation.

Stage of Research

The inventors have devised a new method for the in-situ detection of RNA in cells that overcomes several technical challenges of other currently available methods. Specifically, this method does not require the use of microfluidics droplets to physically separate single cells, nor does it require the use of reverse transcriptase. Briefly, a pair of polynucleotides probes is annealed to sequences in target RNA. Subsequently, these sequences are ligated, and a cell-specific barcode is then synthesized in the cell using split-pool rounds. This in turn adds the barcode sequences to the ligated probe pair sequences.

Therefore, the multiple rounds of split-pooling result in ligated probe pair sequences with unique barcodes in each cell. These methods can be performed in fixed and permeabilized cells.

Applications