The workflow incorporates Illumina-specific adaptor sequences during cDNA amplification for the generation of indexed libraries that are ready for sequencing on Illumina platforms. The application also enables you to perform differential expression analysis by 5' end capture in parallel. Please note that the workflow requires a different chip and collection kit depending on whether it is being performed on the SMARTer ICELL8 Single-Cell System or the SMARTer ICELL8 cx Single-Cell System (coming soon). See the Components section for details on the necessary products for each workflow.
Single T cells isolated with the SMARTer ICELL8 systems, as well as positive and negative control samples, are dispensed into a 5,184-nanowell TCR chip that contains preprinted barcodes. Each TCR chip contains 1,728 unique barcodes, and each barcode is printed three times on the chip. Following identification of single-cell-bearing wells using CellSelect Software, SMART RT-PCR reagents are dispensed to the wells of samples chosen for further processing. Barcoded cDNA from the selected wells are PCR-amplified in-chip. The full-length barcoded amplicons are then pooled off-chip, and the purified cDNA is used as template in the first TCR-specific PCR. The product from the first TCR-specific PCR is used as a template in a second TCR-specific PCR to incorporate Illumina indexed primers.