Introduction

The Illumina Single Cell 3′ RNA Prep, T20 kit in combination with the Illumina Single Cell Enrichment and Amplification (SEA) Kit facilitates custom single-cell RNA-Seq applications at the 20,000 cell scale for up to 4 samples per kit.

A common application of single-cell RNA sequencing is the capture of synthetic polyadenylated DNA tags that have been introduced to label select targets in a sample. The synthetic tags used and the methods for labeling unique samples with synthetic tags depends on your needs. The SEA Kit enables the amplification and isolation of these synthetic nucleotide tag (SNT) products to generate sequencing-ready libraries compatible with Illumina Single Cell 3′ RNA Prep.

Starting from single cell suspensions, the Illumina Single Cell Supplemental Enrichment and Amplification Kit uses particle-templated instant partitions (PIPs) to combine labeled cells with barcoded beads to produce sequencing-ready libraries in less than 2 days. These libraries enable 3ʹ gene expression profiling, multiplexed cellular protein measurements, and sample multiplexing of 80,000 individual cells per kit.

Illumina Single Cell 3′ RNA and SEA Kit libraries can be pooled together for sequencing, taking into account the differences in cell number and read depth requirements for each library type. Sequencing depth varies based on your application needs, but starting recommendations are listed in the following table.

Number of Synthetic Tags	SNT Reads per Input Cell	Sequencing Pooling Ratios (SNT:Illumina Single Cell 3′ RNA)
1	500	1:40
< 100	5,000	1:4
> 100	10,000	1:2

These libraries require a minimum of 1% PhiX to be added to the loaded library pools. It is recommended to increase PhiX to a minimum of 2% for NovaSeq X Series sequencing instruments.