Single-Cell Transcriptome Library Construction via In-Situ Reverse Transcription

The patent applies preliminary action by performing in-situ reverse transcription within intact cells before droplet encapsulation. This allows cDNA synthesis to occur while cells are still in their native state, eliminating the need for post-encapsulation lysis and reducing steps required to avoid cell doublets. The reverse transcription is performed in the well plate format with indexed oligo-dT primers added to each well, enabling pre-indexing of cells before droplet generation.

The patent segments the sequencing process into distinct phases: in-situ reverse transcription in well plates with individual indexing, followed by droplet encapsulation for PCR amplification. This segmentation allows each phase to be optimized independently - the reverse transcription phase can handle high cell densities in parallel wells, while the droplet phase focuses on amplification without the constraint of avoiding doublets during the indexing step.

The patent performs preliminary indexing of cells through in-situ reverse transcription with indexed oligo-dT primers before droplet encapsulation. This pre-indexing allows the system to tolerate higher cell densities during droplet generation because the barcoding information is already established. The indexed primers are added to each well at controlled concentrations, ensuring that even if multiple cells are present in a droplet, each cell's transcripts are already tagged with well-specific barcodes that can be distinguished during analysis.

The patent introduces an intermediary indexing step using indexed oligo-dT primers that serve as mediators between the cell population and the droplet encapsulation process. These primers carry barcodes that identify the well of origin, acting as an intermediary layer that decouples the cell density control from the single-cell identification accuracy. This allows high cell densities to be processed while maintaining the ability to distinguish individual cell transcripts through the barcode information.

The patent merges the reverse transcription and indexing steps into a single in-situ operation performed within the same well plate format. Instead of separate manual indexing steps followed by cell recovery and redistribution, the indexed oligo-dT primers are added directly to each well containing the cells, and reverse transcription occurs in place. This combining of steps eliminates the need for multiple cell recovery and redistribution operations, reducing both device complexity and operational burden while maintaining high throughput.

The indexed oligo-dT primers serve multiple functions simultaneously: they prime reverse transcription of polyadenylated transcripts, they provide barcodes for cell identification, and they enable magnetic bead capture of the first-strand cDNA. This multi-functionality eliminates the need for separate reagents and steps for each function, simplifying the overall experimental workflow while achieving high-throughput indexing of thousands of cells in parallel wells.

The patent performs preliminary reverse transcription within intact cells in the well plate before any cell recovery or manipulation steps. The indexed oligo-dT primers hybridize to poly A tails of mRNA and initiate cDNA synthesis while cells remain in their original wells. This preliminary action ensures that indexing is completed before cells are subjected to recovery procedures, eliminating the risk of losing already-indexed cells during redistribution steps and preventing loss of substance while maintaining manufacturing precision.

The in-situ reverse transcription system is designed to work within the cells themselves without requiring cell lysis or recovery. The indexed oligo-dT primers and reverse transcriptase reagents are added directly to each well containing the cells, and the reverse transcription reaction proceeds in place. The cells essentially serve their own function as reaction vessels, eliminating the need for external recovery and transfer steps that would cause cell loss, thereby reducing substance loss while maintaining precise indexing.