SPLiT-seq – single-cell profiling with split-pool barcoding

To facilitate scalable profiling of single cells, engineers at the University of Washington have developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing and requires no customized equipment. The developers used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. Over 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation. Pseudotime analysis revealed transcriptional programs driving four developmental lineages, providing a snapshot of early postnatal development in the murine central nervous system. SPLiT-seq provides a path toward comprehensive single-cell transcriptomic analysis of other similarly complex multicellular systems.

Overview of SPLiT-seq

(A) Labeling transcriptomes with split-pool barcoding. In each split-pool round, fixed cells or nuclei are randomly distributed into wells and transcripts are labeled with well-specific barcodes. Barcoded RT primers are used in the first round. Second and third round barcodes are appended to cDNA through ligation. A fourth barcode is added to cDNA molecules by PCR during sequencing library preparation. The bottom scheme shows the final barcoded cDNA molecule. (B) Species mixing experiment with a library prepared from 1,758 whole cells. Human UBCs are blue, mouse UBCs are red, and mixed-species UBCs are gray. The estimated barcode collision rate is 0.2%, whereas species purity is >99%. (C) UMI counts from mixing experiments performed with fresh and frozen (stored at -80°C for 2 weeks) cells and nuclei. Median human UMI counts for fresh cells: 15,365; frozen cells: 15,078; nuclei: 12,113; frozen nuclei: 13,636. (D) Measured gene expression by SPLiT-seq is highly correlated between frozen cells and cells processed immediately (Pearson-r: 0.987). Frozen and fresh cells were processed in two different SPLiT-seq experiments.