Additional file 1: Figure S1. of Cell fixation and preservation for droplet-based single-cell transcriptomics

Computational cell selection and RNA, cDNA library and cell quality. Related to Fig. 2. (a) Identification of cell barcodes associated with single-cell transcriptomes in a pool of amplified single-cell libraries. Drop-seq involves Poisson-limited dilution of cells, implying that most beads (> 95%) are only exposed to ambient RNA. To identify the cell barcodes associated with cellular transcriptomes, cell barcodes are plotted in decreasing order of reads against the cumulative fraction of reads. The inflection point (red line) indicates the number of cells; human-mouse cell doublets were removed computationally. Note that sample 'Fixed 1 week' has fewer cells, because only a fraction of barcoded beads was used for library preparation. (b) A subset of cells from the experiment depicted in Fig. 2 (Live: 99 human, 44 mouse cells; Fixed: 253 human, 90 mouse cells) was sequenced at a higher median depth of ~104,106 and ~53,500 aligned reads per cell. Note that the live sample appears to have more genes and UMIs, because fewer cells were sequenced, resulting in more reads per cell. (c)–(e) Bioanalyzer traces. (c) High-quality RNA could be extracted from rehydrated cells that were fixed and stored for 20 weeks. (d) Fixation and storage does not change the fragment size distribution of Drop-seq cDNA libraries. Libraries were purified with 0.6× Solid Phase Reversible Immobilization (SPRI) beads. (e) Parallel control purification of the cDNA library ’Fixed 3 weeks’ with 0.6× (fragments above 500 bp; upper panel) or 1.8× SPRI beads (all fragments; lower panel) did not reveal a major peak corresponding to small molecular weight fragments indicative of low-quality RNA input cells. (f) Plot depicting the percentage of reads mapping to non-mitochondrially encoded genes. Stressed or broken cells lose non-mitochondrially encoded, cytoplasmically localized mRNAs [16]. Loss of cytoplasmic reads in fixed cells was < 10%. (PDF 481 kb)