The development of high-throughput single-cell RNA sequencing (scRNA-seq) has enabled access to information about gene expression in individual cells and insights into new biological areas. Although the interest in scRNA-seq has rapidly grown in recent years, the existing methods are plagued by many challenges when performing scRNA-seq on multiple samples. To simultaneously analyze multiple samples with scRNA-seq, Yonsei University researchers developed a universal sample barcoding method through transient transfection with short barcode oligonucleotides. By conducting a species-mixing experiment, the researchers have validated the accuracy of their method and confirmed the ability to identify multiplets and negatives. Samples from a 48-plex drug treatment experiment were pooled and analyzed by a single run of Drop-Seq. This revealed unique transcriptome responses for each drug and target-specific gene expression signatures at the single-cell level. This cost-effective method is widely applicable for the single-cell profiling of multiple experimental conditions, enabling the widespread adoption of scRNA-seq for various applications.
(A) Scheme of multiplexed scRNA-seq by transient barcoding method using SBOs. (1) Samples with various conditions are prepared. (2) Each sample is transfected with SBO containing a unique sample barcode. (3) Barcoded cells are pooled together and processed for scRNA-seq (e.g., Drop-Seq). (4) Cells are lysed within droplets, and the released mRNAs and SBOs are captured, reverse-transcribed, and sequenced. (5) Cells are demultiplexed and assigned to their origins and processed for further analysis. (B) Heatmap of normalized SBO counts for 6-plex human/mouse species-mixing experiment. Rows represent cells, and columns represent SBOs. Cells are assessed whether they are positive for a particular SBO based on the SBO count matrix (see Materials and Methods). Cells were classified as singlets (positive for a unique SBO), multiplets (positive for more than one SBO), or negatives (not positive for any SBO) and ordered by their classifications. (C) Scatter plot showing raw counts between two SBOs. SBOs 1 and 6 were used to barcode different samples (Human 1, Mouse 2) (left). SBOs 3 and 4 were used to barcode the same sample (Human 3) (right). (D) Species-mixing plot of samples associated with SBOs 1 and 5. Cells were labeled according to their SBO classifications. Black dots indicate Human 1 sample barcoded with SBO 1, red dots indicate Mouse 1 sample barcoded with SBO 5, and gray dots indicate doublets that are positive for both SBOs. (E) Distribution of RNA transcript counts in cells between singlets (green), multiplets (blue), and negatives (red). Negatives, which imply beads exposed to ambient RNA, had the lowest number of transcripts. Multiplets had slightly more transcripts than singlets, indicating more RNA content within a droplet.