Custom T7-polyA primers (N10B1 → N10B16, Integrated DNA Technologies, USA) were designed containing 9bp error correcting sequences for identifying single cells (sample barcode) and 10bp random nucleotide sequences (UMI/varietal tag) to label each mRNA molecule amplified with a unique barcode. The UMI allows for elimination of reads containing duplicate tags for the same mapped sequence and only tally up the total unique tags of all mapped sequence to a coding sequence. This primer also contained a 26bp flanking RA5 adapter sequence needed for downstream Illumina cDNA library step, which eliminates a rate limiting enzymatic 5′ ligation step of cDNA preparation increasing efficiency (Hashimshony et. al. 2012).
