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. 2020 Dec 21;117(52):33404–33413. doi: 10.1073/pnas.2010738117

Fig. 1.

Fig. 1.

HyPR-seq enables single-cell quantification of selected RNAs. (A) At a probe binding site, initiator probes bind adjacent to one another on an RNA of interest, creating a binding site for a hairpin oligo. A final readout oligo is annealed and ligated, forming a template for PCR amplification containing transcript information, UMI, and handles for PCR and sequencing. (B) Experimental workflow for HyPR-seq. (C) Comparison of three single-cell methods in K562 cells. (Top) HCR measurements of GAPDH and GATA1 using probes targeting the same binding sites used for HyPR-seq. (Middle) Histogram of UMIs per cell for three probes targeting GAPDH and GATA1 from HyPR-seq. (2,148 cells) and (Bottom) 10X Genomics Chromium 3′ scRNA-seq (207,324 cells). (Scale bar, 10 μm.) All experiments performed in K562 cells. (D) HyPR-seq counts per cell for each of the three probes targeting GAPDH (left columns), GATA1 (center columns), or GFP (not expressed, right side). Error bars represent 95% confidence interval (CI) of the mean from two replicates (from 4,605 cells). For more analysis of probe variability, See SI Appendix, Fig. S4D. (E) Counts per cell at a constant depth of 1,000 total UMIs per cell for three genes (GATA1, PGK1, and TUBB) as assayed by 10X Genomics Chromium 3′ scRNA-seq (26) and HyPR-seq. Counts for HyPR-seq are shown for the 22-gene experiment in K562 cells and 2 simulated experiments (based on these counts) measuring different numbers of total genes. Horizontal bars show the caps of the (vertical) error bars, which represent 95% CI of the mean from two replicates. (F) Counts per cell for 19 genes in K562 cells as measured by 10X Genomics Chromium 3′ scRNA-seq (x axis) and HyPR-seq (y axis). Error bars for HyPR-seq measurements show 95% CI of the mean from two replicates (4,605 total cells). (G) In THP1 cells ± LPS, fold changes of 18 genes assayed by bulk RNA-seq (x axis) and HyPR-seq (y axis). Error bars represent 95% CI of the mean from three (RNA-seq) or four (HyPR-seq) replicates (13,957 total cells). (H) To assess mixing, K562 cells were transduced to express two different barcode RNAs, each detectable by three HyPR probes. Scatterplot shows UMI counts for each barcode per droplet (2,727 total droplets). Unassigned cells are in gray.