Protocol for high-resolution multiplexed sequencing of single-cell full-length transcriptome with CBTi-seq

Balancing throughput and full-length coverage remains challenging in single-cell RNA sequencing (scRNA-seq). Here, we present combinational barcoded Tn5 transposon insertion sequencing (CBTi-seq), a highly scalable protocol for constructing multiplexed, full-length transcriptome libraries from single cells and micro-region tissues. The workflow details sample acquisition, one-step reverse-transcription PCR (RT-PCR) cDNA amplification, orthogonal combinational barcoded Tn5 tagmentation, multiplexed pooling, dual-option purification, and final library enrichment. For complete details on the use and execution of this protocol, please refer to He et al.¹.