Experimental design [29-31] |
Organism | Culture, animal, human |
Replicates/number of cells | General considerations including organism heterogeneity and ability to resolve rare cell populations of interest |
Tissue disaggregation | Mechanical dissection, enzymatic disaggregation |
Enrichment | FACS, magnetic bead separation, centrifugation |
Cell capture | |
Microfluidics | |
Nanowell [32, 33] | |
Droplet [34] | Advantages in throughput and cost |
FACS | Relatively lower throughput; protein markers needed |
Plate-based | |
Micropipette/laser capture microscopy [7, 35] |
Library preparation [36] | NGS Illumina versus other cDNA-compatible system; availability depends on protocol |
3' | Does not capture splice variants; lower sequencing depth required |
5' | Requires sequencing to higher read depth |
Full-length transcript | Costly; splice variant, isoform and allelic variation analysis |
Quality | |
Cell integrity | |
FACS | Sorting out dead cells, cell fragments |
Imaging | Plate-based systems; detection of empty wells, doublets |
RNA quality | |
RIN [37] | RNA quality score |
Barcoding | |
Cell label | One sequence per bead/well |
UMI [38] | Different for every oligo on bead; differentiates PCR clone from transcript read |
Multiplexing | |
RNA spike-in | |
Animal doping | Multiplexing with cells of different species, detect doublets |
Multiple donor [39, 40] | Using SNPs from unrelated donors or oligo-tagged antibodies to detect doublets, correct for batch effect in multiplexed experiments |