Shielded Barcoded RNA for Multiplex Single-Cell Sequencing
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Solution Overview
Problem
High-throughput parallel profiling of multiple samples in single-cell RNA sequencing is challenging due to limitations such as library preparation cost and batch effects, and existing methods like antibody-based labeling are limited by the availability of cell surface markers, making pre-pooling experiments infeasible, especially for in vivo studies.
Innovation Solution
Development of barcoded RNA constructs with shield sequences, barcode sequences, scaffold sequences, and capture sequences, which can be introduced to cells using viral vectors, allowing for multiplexing and demultiplexing of samples, enabling efficient single-cell RNA sequencing and gene expression profiling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If antibody-based labeling is used for multiplex single-cell RNA sequencing, then cell surface markers can be labeled and pooled, but the method is limited by the availability of cell surface markers and requires immediate pooling before loading
Solution Approach 1:
The patent applies preliminary action by introducing barcoded RNA constructs into cells before sampling and pooling. The barcodes are pre-installed in the cells' transcriptome, allowing samples to be pooled at any time without immediate processing requirements. This resolves the timing constraint of antibody-based methods where pooling must occur immediately before loading.
2Adaptability or versatility
If polyA labeling using lentiviruses is used for intracellular genetic barcoding, then cell barcodes can be recovered from mRNA library, but the entire expression cassette must be designed in reverse orientation and extra polyA signals added
Solution Approach 1:
The patent applies inversion by designing the expression cassette in the forward (sense) orientation rather than the reverse (antisense) orientation required by conventional polyA labeling methods. The barcoded RNA construct is transcribed from a sense-oriented promoter, eliminating the need for reverse orientation design and extra polyA signals, thereby simplifying the expression cassette design while maintaining intracellular barcoding functionality.
3Measurement precision
If conventional barcoding methods are used, then cell identification is possible, but additional dial-out PCR steps are required to recover barcodes
Solution Approach 1:
The patent applies extraction by incorporating a capture sequence into the barcoded RNA construct that specifically binds to magnetic beads. This allows direct isolation and recovery of barcoded RNA from total cellular RNA without requiring additional dial-out PCR steps. The capture sequence enables selective extraction of barcoded molecules, improving both productivity by eliminating PCR steps and maintaining measurement precision through specific binding.
4Productivity
If high-throughput parallel profiling is performed, then transcriptional profiling of thousands of cells is achieved, but library preparation cost and batch effects increase
Solution Approach 1:
The patent applies merging by combining multiple samples with different barcoded RNA constructs into a single pooled library for simultaneous sequencing. All samples undergo identical library preparation and sequencing procedures, eliminating batch effects between separate processing runs. The unique barcodes allow computational demultiplexing of the pooled data, maintaining sample identity while achieving high-throughput parallel profiling with improved reliability.
Data Source
AI summary
The present disclosure relates to a barcoded RNA comprising a first shield sequence at the 5′ end of the barcoded RNA, a barcode sequence, a scaffold sequence, a capture sequence, and a second shield sequence at 3′ end of the barcoded RNA. The present disclosure also provides for methods of performing single-cell RNA sequencing using the barcoded RNA. The present disclosure also provides for libraries including the barcoded RNA.