STRT-seq-2i: dual-index 5' single cell and nucleus RNA-seq on an addressable microwell array

Abstract

Single-cell RNA-seq has become routine for discovering cell types and revealing cellular diversity, but archived human brain samples still pose a challenge to current high-throughput platforms. We present STRT-seq-2i, an addressable 9600-microwell array platform, combining sampling by limiting dilution or FACS, with imaging and high throughput at competitive cost. We applied the platform to fresh single mouse cortical cells and to frozen post-mortem human cortical nuclei, matching the performance of a previous lower-throughput platform while retaining a high degree of flexibility, potentially also for other high-throughput applications.

Figure 1

(a) STRT-seq-2i workflow overview. (b and c) Distribution of molecule (b) and gene counts (c) for cortex data (Fig. 2). (d) Coefficient of variation (CV) as a function of mean number of molecules m expressed in cortex cells. The fitted line represents an offset Poisson, $Lo{g}_{2}CV=Lo{g}_2(m^{-0.5}+0.5)$. (e and f) Doublet rates as estimated by the ratio of species-specific molecules, per well, in mouse-human (e) and chicken-human (f) two-species experiments. (g and h) Single-cell well success rate when addressing 100 wells per unit by (g) limiting dilution or (h) FACS with 200 nL (left) or 50 nl PBS (right) predispensed. (i) Accuracy of FACS demonstrated by checkerboard pattern sort to 32 wells per unit.

Figure 2

Heterogeneity of cell-types in the mouse somatosensory cortex and human temporal cortex. (a) tSNE visualization for clustering of 2192 single-cells, colored by BackSPINv2 clusters. (b) Top marker genes of each cell type presented as normalized average expression by cluster, with major cell classes overlayed by colored boxes. (c) Genes specific to pyramidal neuron subclasses by layer specificity, confirmed by in situ hybridization from Allen Mouse Brain Atlas. Image credit: Allen Institute. (d) tSNE visualization for clustering of 2028 post-mortem isolated neuronal nuclei from the middle temporal gyrus, colored by BackSPINv2 clusters. (e) Top marker genes of each neuronal subtype presented as normalized average expression by cluster. (f) Validation of pyramidal neuron (Glut) gene expression layer specificity, by in situ hybridization from Allen Human Brain Atlas. The outermost layers I and VI are indicated by strokes. Image credit: Allen Institute.