By leveraging Takara Bio"s expertise in single-cell RNA-seq library construction and the cell isolation and selection capabilities of the ICELL8 platform, the ICELL8 cx 3" DE workflow enables high-throughput preparation of Illumina libraries for differential expression analysis of single cells from up to eight different samples in parallel.

The ICELL8 cx 3" DE workflow for the ICELL8 cx Single-Cell System uses the ICELL8 3" DE Reagent Kit (Cat. # 640167); the ICELL8 cx 3" DE Chip (Cat. # 640199), a 5,184-nanowell SmartChip that contains preprinted oligo(dT) primers, and reagents for cDNA synthesis and Illumina library prep; the ICELL8 cx Loading Kit (Cat. # 640197); and the ICELL8 Collection Kit – L (Cat. # 640212)—all purchased separately.

Following staining and dilution, sample cells are dispensed into chip nanowells using the MultiSample NanoDispenser (MSND) part of the cx system. Wells containing single, viable cells are selected using the ICELL8 cx Imager and the ICELL8 cx CellSelect Software provided with the ICELL8 cx system. Following the selection of cells for processing and analysis, RT-PCR reagents are dispensed into the corresponding wells, and cDNA synthesis and amplification are performed in-chip in a single run. Following RT-PCR, cDNA products are pooled and purified, and sequencing libraries are prepared using a transposase-based method that enriches for cDNA derived from mRNA 3" ends, followed by a limited-cycle PCR amplification step that incorporates Illumina index sequences.

Sequencing data from the resulting libraries can be traced back to individual cells using the nanowell-specific barcode sequences and can be used to perform differential gene expression analysis by mRNA 3" end-counting. By yielding high-quality, 3"-focused sequencing data from hundreds of cells in parallel, the kit provides an efficient, cost-effective solution for high-throughput differential expression analysis of single cells.