Supplemental Enrichment and Amplification (SEA) primer design for Illumina Single Cell RNA Kits

General Guidance across all applications

The Supplemental Enrichment and Amplification (SEA) kit can amplify any polyadenylated synthetic nucleotide tag (SNT). A SNT is any polyadenylated reporter molecule such as probes for surface epitope labeling of proteins, or hashtag oligonucleotides used for pooling multiple samples into a single reaction. The use of the SEA kit with third party products for cell surface labeling and cell hashing applications has been customer demonstrated using community-verified protocols. However, Illumina does not have any recommendation or partnership for these third party products. Labs must independently determine whether using any third-party solutions with the SEA kit is appropriate for their workflow or laboratory and whether a third-party license is required.

The forward primer for all supplemental enrichment and amplification reactions must be conserved in order to retain the single-cell barcode structure in the PCR product. Meanwhile, the reverse primer is tailored to the customer’s application. Generally, it is best practice to get experience with the base Illumina Single Cell 3' RNA Prep kit workflow before increasing workflow complexity with the SEA kit. See The importance of pilot studies for Illumina Single Cell RNA Preparation prior to workflow changes for more information. All sequences listed in this document are listed 5' to 3'.

Note that the WTA-F-V2 primer is a 21bp truncated TruSeq Read 1 sequence.

The Tm of WTA-F-V2 is 69°C demonstrated empirically with PIPs present during the PCR amplification reaction and for all SEA kit applications. If labs are using a primer design application to determine the Tm of their reverse primer for their SEA kit application, such as SnapGene or Benchling, the Tm of WTA-F-V2 is 67°C in silico and the reverse primer should be matched to 67°C in silico.

Figure 1. Shows the on-bead binding site of the WTA-F-V2 forward primer in the Illumina Single Cell Final Library Structure

Oligonucleotides are required but not provided in the Illumina kits. Illumina recommends ordering primers from an oligonucleotide provider. Order DNA oligonucleotide primers at 100 nmol scale with desalted purification. If preferred, order primers resuspended to a stock concentration of 100 µM in 1X IDTE Buffer. If ordering lyophilized primers, upon receipt resuspend the primers to a stock concentration of 100 µM in 1X IDTE Buffer. When ordering, include phosphorothioate internucleoside linkers, commonly coded as asterisks, between the last three base pairs of primers for increased primer stability.

When combining two dial-out use cases into a single SEA kit amplification, it is recommended to validate primer design by doing single use-case experiments first before combining applications into a single reaction. In the context of this article, a "dial-out" is the round 1 PCR targeted amplification using a SEA kit reverse primer designed by and supplied by the customer. These dial-out amplifications will undergo library prep with Illumina sequencing compatible Unique Dual Indexes (UDIs) for sequencing on an Illumina sequencing instrument.

Synthetic Nucleotide Tag (SNT) or Hash Tag Oligo (HTO) Applications

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 the needs of the customer. The reverse primer customization for the dial-out PCR will require an additive primer that matches the PCR-handle of the commercially available Synthetic Nucleotide Tag (Figure 2; Purple Region) or Hashtag Oligonucleotide (Figure 3; Orange Region) purchased from a third party vendor. This application requires the order of custom sample indexing primers where the 3' end of the indexing primer must match the PCR handle of the commercially available SNT or HTO.

Figure 2.Shows the functional group reverse primer design for SNT applications where the Purple Region is the SNT PCR handle for round 1 PCR and where TruSeq Read 2 is used for the i7 in SNT Library indexing PCR.

Figure 3. Shows the functional group reverse primer design for HTO applications where TruSeq Read 2 is the PCR handle on the HTO used for the reverse primer design for round 1 PCR and the i7 for HTO Library indexing PCR.

The functional groups of the Oligonucleotides for SNT amplification in the SNT User Guides will be covered here:

5'- SNT PCR handle - 3'

5'- TruSeq Read 2 PCR handle of HTO - 3'

Warning: SNT and HTO use cases require user-supplied custom sample-indexing primers!

The functional groups of the Oligonucleotide Indices for SNT indexing will be covered here:

For the X's refer to the Illumina Single Cell RNA Index (96 samples) i5 and i7 adaptor sequences and be sure to avoid SCUDP0017 thru SCUDP0024 as these are the sequences provided in the Illumina Single Cell 3' RNA Prep Index Strip in the standard Illumina Single Cell 3' RNA prep kits. For proper color balancing refer to the Illumina Index adaptors pooling guide for the Illumina Single Cell Unique Dual Indexes (96 indexes, 96 samples) plate (20132788) and the Illumina Single Cell Prep UDI Mix Strip.

Note that Additive Primer's A and H may need different sequences if the PCR handles of the commercially available polyadenylated SNT of choice does not use the sequences presented here. Otherwise if following the above strategy, custom indexing primers need to be ordered with the structure in the table above.

CROPseq sgRNA Applications

Use the suggested oligonucleotide sequences to amplify the bead-bound cDNA that are generated from the captured sgRNAs. Alternatively, labs can use the oligonucleotide sequences to generate sequencing-ready libraries for Illumina sequencing with Illumina Single Cell 3′ RNA kits. To amplify cDNA complementary to the sgRNAs, an sgRNA-specific reverse primer targeting the conserved U6 site of the gRNA's is added to the PCR amplification reaction alongside the standard WTA-F-V2 forward primer. Note that it is recommended to use the PE2 Nextera sequence at the 5’ end of the reverse primer to make the PCR dial out compatible with the Illumina Single Cell Unique Dual Indexes (96 indexes, 96 samples) plate (20132788). Be sure to avoid the use of column number 3 as those sequences (SCUDP0017 thru SCUDP0024) are provided in the 3’ Illumina Single Cell kits in the Illumina Single Cell Prep UDI Mix Strip. For color balancing refer to the Illumina Index adaptors pooling guide for the Illumina Single Cell Unique Dual Indexes (96 indexes, 96 samples) plate (20132788) and the Illumina Single Cell Prep UDI Mix Strip.

Warning: Make sure to use separate unique UDIs for the mRNA gene expression library created using the Illumina Single Cell 3' RNA Prep kit from the UDI's used for the CROPseq sgRNA library constructed using the SEA kit.

Figure 4. Shows the functional group reverse primer design for sgRNA CROPseq applications with the sgRNA U6 site shown in dark blue and the PE2 Nextera PCR handle shown in orange

Here is a suitable sgRNA construct example:

CROPseq sgRNA Reverse Primer Design

Note that the sgRNA U6 sequence may be different than this example if using a different CROPseq vector.

The Tm of WTA-F-V2 is 69°C demonstrated empirically with PIPs present during the PCR amplification reaction and for all SEA kit applications. If labs are using a primer design application to determine the Tm of their reverse primer for their SEA kit application, such as SnapGene or Benchling, the Tm of WTA-F-V2 is 67°C in silico and the reverse primer should be matched to 67°C in silico.

Other custom application guidance

If doing a custom application such as a targeted gene dial-out amplification, note that it is recommended to use the PE2 Nextera sequence at the 5’ end of the reverse primer to make the PCR dial out compatible with the Illumina Single Cell Unique Dual Indexes (96 indexes, 96 samples) plate (20132788). Be sure to avoid the use of column number 3 as those sequences are provided in the 3’ Illumina Single Cell kits in the Illumina Single Cell Prep UDI Mix Strip. For proper color balancing refer to the Illumina Index adaptors pooling guide for the Illumina Single Cell Unique Dual Indexes (96 indexes, 96 samples) plate (20132788) and the Illumina Single Cell Prep UDI Mix Strip.

There is no official user guide for this application. Users are responsible for optimizing PCR conditions and magnetic bead clean-up ratios for their custom target amplicon workflows.

Warning: This method will provide read counts only and no molecular/transcript counts, so customers should assess if this approach is appropriate for their experimental needs.

Warning: Make sure to use separate unique UDI's for the mRNA gene expression library created using the Illumina Single Cell 3' RNA Prep kit from the UDIs used for the targeted gene library constructed using the SEA kit.

Figure 5.Shows the functional group reverse primer design for custom targeted gene applications with the Custom Targeted gene sequence in dark blue and the PE2 Nextera PCR handle shown in orange.

Custom Reverse Primer Design

Note: Xs represent customer designed targeted region that must be appended.

The Tm of WTA-F-V2 is 69°C demonstrated empirically with PIPs present during the PCR amplification reaction and for all SEA kit applications. If labs are using a primer design application to determine the Tm of their reverse primer for their SEA kit application, such as SnapGene or Benchling, the Tm of WTA-F-V2 is 67°C in silico and the reverse primer should be matched to 67°C in silico.

The below PCR profile can be used as a starting point for optimization for custom target gene amplicon experiments.