By leveraging Takara Bio's expertise in single-cell RNA-seq library construction and the cell isolation and selection capabilities of the SMARTer ICELL8 system, the SMARTer ICELL8 Chip and Reagent 3' DE Kit enables high-throughput preparation of Illumina libraries for differential expression analysis of single cells from up to eight different samples in parallel. Included in the kit are a SMARTer ICELL8 Chip v2, a 5,184-nanowell SmartChip that contains preprinted oligo(dT) primers, and reagents for cDNA synthesis and Illumina library prep. For researchers interested in using unique molecular identifiers (UMI) for 3' differential expression experiments, we offer the SMARTer ICELL8 Chip and Reagent Kit v2 which also includes SMARTer ICELL8 Chip v2 and the necessary library prep reagents.
Following staining and dilution, sample cells are dispensed into chip nanowells using the ICELL8 MultiSample NanoDispenser (MSND), and wells containing single, viable cells are selected using CellSelect software provided with the SMARTer ICELL8 system. Following 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 produced by either kit 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, these kits provides an efficient, cost-effective solution for high-throughput differential expression analysis of single cells.