Product Applications

The Illumina Single Cell 3′ RNA protocol with supplemental enrichment of SNTs requires a suspension of high-quality single cells that are labeled with polyadenylated SNTs. These labeled cells are then input into the Illumina Single Cell 3′ RNA Prep, T2 Kit to enable sequencing of exogenous SNT libraries alongside gene expression.

To begin, use a preferred protocol (not provided) to label the cells or cellular proteins of interest with polyadenylated SNTs. Input the labeled cells into the Illumina Single Cell 3′ RNA Prep, T2 Kit to generate bead-bound single-stranded cDNA via reverse transcription of captured target mRNA. This add-on protocol provides instructions for amplification and isolation of exogenous SNTs captured in the Illumina Single Cell 3′ RNA workflow alongside gene expression. To amplify the mRNA-derived cDNA, follow the Illumina Single Cell 3′ RNA Prep, T2 Kit Product Documentation through recovery of the 3' mRNA cDNA, and then use the remaining PIPs pellet to amplify SNT-derived cDNA. The SNT-derived cDNA is then used as input for library preparation. Illumina recommends specific oligonucleotide sequences for samples labeled with common SNTs, but you can adapt this protocol for any desired exogenous SNT.

For custom applications, append a PIPseq-compatible SNT sequence to the desired target. The sequence must contain TruSeq Read 2 sequences to ensure compatibility with the Illumina Single Cell 3′ RNA primer sets in Oligonucleotides. The following example shows a compatible SNT sequence:

(5′-CCTTGGCACCCGAGAATTCCACATGATTGGCTCBAAAAAAAAAAAAAAAAAAAAAAAAAAAA*A*A -3′)