Droplet microfluidics based combinatorial indexing for massive-scale 5′-end single-cell RNA sequencing

Abstract Single-cell RNA sequencing methods focus on 5’-end of transcripts can reveal promoter and enhancer activity and allows efficiently profiling immune receptor repertoire. However, ultra-high-throughput 5′-end single-cell RNA sequencing methods have not been described. Here, we introduce five-prime end single-cell combinatorial indexing RNA-Seq (FIPRESCI), enabling the massive sample multiplexing and increasing the throughput of the droplet-microfluidics system by over 10-fold. FIPRESCI is based on combining the ability of indexed Tn5 transposome to barcoding RNA/cDNA hybrids heteroduplexes in situ and Template Switching Oligo barcoding of commercial droplet-microfluidic. Using FIPRESCI, we profiled transcriptome and transcribed cis-regulatory elements from various human and mouse cell lines and demonstrated the approach is compatible for both cells and nuclei. We applied FIPRESCI to E10.5 whole mouse embyro and uncover previously unknown isoform switch during GABAergic neurogenesis of several important regulators, including Rbfox2. We further applied FIPRESCI to primary T cells from human peripheral blood mononuclear cells (PBMCs) and demonstrated it enable simultaneous identification of cancer specific gene expression and T cell receptor (TCR) signatures. Given its simplicity, flexibility, and scalability, FIPRESCI will have wide application in cell atlas projects, large-scale screening, and single-cell immune profiling of large cohorts’s studies.