Extended Data Fig. 4: scifi-RNA-seq yields high-quality data for whole cells, fresh nuclei and fixed nuclei.

a, Performance metrics for scifi-RNA-seq experiments using a mixture of human Jurkat cells and mouse 3T3 cells, starting from whole cells permeabilized by methanol, freshly isolated nuclei, and nuclei fixed with 1% or 4% formaldehyde (cryopreserved, re-hydrated, and permeabilized). The following plots are shown: (i) ranked barcodes plotted against reads, unique molecular identifiers (UMIs), and detected genes, distinguishing single-cell transcriptomes from background noise; (ii) reads plotted against UMIs; (iii) reads plotted against the number of detected genes; (iv) reads plotted against the fraction of unique reads; (v) species mixing plot showing the number of UMIs per cell aligning to the mouse genome (x-axis) versus the human genome (y-axis). To facilitate comparisons between the different types of input material, the axes of the performance plots use the same scale across conditions. b, In a species mixing experiment with pre-indexed nuclei from human (Jurkat) and mouse (3T3) cells run at the maximum loading concentration of the standard Chromium protocol (15,300 nuclei per channel), the microfluidic round2 barcode (left plot) is sufficient to resolve single cells. Nevertheless, the combination of round1 and round2 barcodes still improves the separation (right plot). c, Coverage along human and mouse transcripts from 200 bp upstream of the transcription start site (TSS) to 200 bp downstream of the transcription end site (TES), shown for whole cells permeabilized by methanol, freshly isolated nuclei, and nuclei fixed with 1% or 4% formaldehyde (cryopreserved, re-hydrated, and permeabilized). Freshly isolated nuclei show the strongest 3’ enrichment. d, Box plots summarizing sequence alignment metrics across the different types of input material: Total reads sequenced, percent uniquely mapped reads, percent multi-mappers, percent alignments to exons plus introns, percent alignments to exons, and percent spliced reads. Freshly isolated nuclei showed the best performance for these alignment metrics. The box plots summarize a total of 2,299 whole cells; 2,000 fresh nuclei; 2,051 nuclei fixed with 1% formaldehyde and 1,896 nuclei fixed with 4% formaldehyde. Box plots depict the interquartile range with marked median and whiskers extending to 1.5 times the interquartile range.