|Instrument||Recommended Number of Samples||Read Length|
|NextSeq 550 System||4 to 6 samples per run (based on a minimum of 100,000 reads per cell)||read 1: 68 bp, read 2: 75 bp|
|HiSeq 2500 System||Rapid run: 4-5 samples per lane; high output v4: 4 samples per lane (based on a minimum of 100,000 reads per cell)||read 1: 68 bp, read 2: 75 bp|
|SureCell WTA 3′ Library Prep Kit for the ddSEQ System||TruSeq RNA Library Prep Kit v2||Illumina Stranded mRNA Prep||Illumina RNA Prep with Enrichment|
|Assay Time||11-12 hours||~10.5 hours||6.5 hours||< 9 hours|
|Content Specifications||Captures the coding transcriptome (3' enriched), with strand-specific information||Captures the coding transcriptome (without strand information)||Captures the coding transcriptome with strand information||Captures the coding transcriptome when used with Illumina Exome Panel|
|Description||Enables single-cell RNA-Seq studies using a droplet generator that isolates ~1200 cells per cartridge, and a simple library prep process.||A simple, cost-effective research solution for analysis of the coding transcriptome.||A simple, cost-effective solution for analysis of the coding transcriptome with precise strand information||A reproducible, economical solution enabling targeted transcript detection and discovery from a broad range of sample types and inputs including formalin-fixed, paraffin- embedded (FFPE) tissues and other low-quality samples|
|Hands-On Time||4-5 hours||~4.5 hours||< 3 hours||< 2 hours|
|Input Quantity||~45,000 cells per cartridge (required input)||0.1 - 1 ug total RNA or 10 - 400 ng previously isolated mRNA (from species with polyA tails)||25-1000 ng standard-quality total RNA||10ng total RNA from fresh/frozen samples, or 20ng total RNA from FFPE samples|
|Mechanism of Action||Oligo dT capture, single cell||Oligo-dT beads capture polyA tails||PolyA capture, ligation-based addition of adapters and indexes||Bead-linked transposome|
|Method||mRNA Sequencing||mRNA Sequencing||mRNA Sequencing||mRNA Sequencing, Target Enrichment, Targeted RNA Sequencing|
|Multiplexing||1-24||Up to 24-plex per lane||Up to 384 Unique Dual Indexes (UDIs)||Up to 384 Unique Dual Indexes (UDIs)|
|Specialized Sample Types||Not FFPE-Compatible, Single Cells||Not FFPE-Compatible||Low-Input Samples, Not FFPE-Compatible||Blood, FFPE Tissue, Low-Input Samples|
|Species Category||Human, Mouse||Bovine, Human, Mammalian, Mouse, Rat||Bovine, Human, Mammalian, Mouse, Rat||Human, Virus|
Mouse cell lines (A20, NIH3T3) and human cell lines (HEK, BJ) were processed using the SureCell WTA 3′Library Prep Kit. Consistently high numbers of detected genes demonstrates that recovery of transcripts is not limited by cell size.
A. BaseSpace-generated plots of the number of unique molecular identifiers (UMI), i.e. transcripts, assigned to the mouse/mm10 (red) and human/hg19 (blue) genomes for each cell barcode. Unique transcripts mapping to both mouse and human (purple) represent cell doublets. B. Cumulated fraction of unique transcripts assigned to cell barcodes (linear scale). The inflection point (red line) determines the number of barcoded cells detected in the sequencing run.
A. PCA clustering of 1384 single cells sorted from a 1:1 ratio mixture of HEK 293 and NIH 3T3 cells, sequenced and analyzed with the BaseSpace SureCell Single-Cell RNA App. B. Cell populations cluster based on expression of the human (hg19) RPL13 gene.
A. Analysis in the BaseSpace SureCell Single-Cell RNA App using the t-SNE algorithm of a mixture of NIH 3T3 and HEK 293 cells identifies a distinct subpopulation of cells. B. Cells color coded by gene expression of hg19 RPL 13 confirms the identity of the subpopulation as human.
Analysis of cell cycle state using the BaseSpace SureCell Single-Cell RNA App is based on unique transcript counts of genes associated with each phase of the cell cycle, normalized by the total count for each cell. Expression is centered by the median and scaled by the median absolute deviation for each cell cycle.