Plate-based 10X Genomics-compatible single-cell RNA-sequencing based on Smart-seq3xpress

TL;DR

A new plate-based method called PB10X improves single-cell RNA-sequencing compatibility with 10X Genomics while offering better performance and cost-effectiveness for small cell populations.

Contribution

PB10X introduces a plate-based, 10X-compatible scRNA-seq strategy that enables indexed sorting and resolves artifacts in existing methods.

Findings

PB10X achieved 81.71% paired TCRαβ chain detection at low sequencing depth.

PB10X detected 4,343 genes and 16,137 UMIs per cell with high read mapping accuracy.

PB10X outperformed SS3X by resolving strand invasion artifacts.

Abstract

10X Genomics (10X) is a leading single-cell RNA-sequencing (scRNA-seq) technology that permits gene expression and T-cell receptor (TCR) repertoire profiling. However, its microfluidics-based approach restricts direct pairing of upstream indexed single-cell sorting with downstream scRNA-seq data and poses challenges for cost-effective profiling of small-scale cell populations.

To address these limitations, we developed a plate-based, 10X-compatible (PB10X) scRNA-seq strategy built on the Smart-seq3xpress (SS3X) principle. PB10X supports indexed sorting using Fluorescence Activated Cell Sorting (FACS) directly into 384-well plates and generates cDNA compatible with any standardized 10X Single Cell 5’ library construction kit including the 5’ V(D)J and 5’ Gene Expression kits. PB10X offers the flexibility of plate-based scRNA-seq with the robustness of the 10X sequencing library preparation, while retaining full compatibility with downstream Cell Ranger data processing.

We demonstrated PB10X’s performance on Jurkat lymphoblasts by generating V(D)J and gene expression sequencing libraries, and benchmarking these against 10X and SS3X. PB10X proved particularly effective for TCR repertoire sequencing, yielding paired TCRαβ chains for 81.71% of the cells at a limited sequencing depth. PB10X further detected a mean of 4,343 genes and 16,137 UMIs per cell, while achieving the highest proportion of uniquely mapped reads and protein-coding genes among the compared methods. Notably, PB10X resolves the strand invasion artifact observed in SS3X.

The novel cost-effective PB10X method offers unprecedented 10X-compatibility in a plate-based format, both for immune receptor repertoire sequencing and gene expression profiling, representing a promising single-cell transcriptomics strategy, especially in immunological settings with limited cell populations.

The online version contains supplementary material available at 10.1186/s12864-025-12286-2.

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