Selecting cycle numbers for High and Low RNA content samples with Illumina Single Cell 3' RNA Prep kits

Assessing cDNA Quality

Illumina Single Cell 3' RNA Prep kits require amplification of a 2 µl aliquot of cleaned up cDNA from the limited 5-cycle PCR reaction of whole transcriptome amplification (WTA) for the Quality Check (QC) of cDNA. This additional amplification is required to assess cDNA quality of samples before libraries are taken to sequencing. The cDNA QC amplification for assessing cDNA quality has two options for QC PCR cycle numbers per kit size dependent on the RNA content of the sample input into the assay.

If the RNA content of a sample is unknown, it is best to adhere to the general recommendations in the reference guides for assessing the cDNA QC PCR cycle number, as shown in Table 1.

• Cultured cell lines should be treated as high RNA content samples and use the lower recommended number of PCR cycles.

• Primary cells isolated from patients or animals as well as nuclei from any sample type should be considered low RNA content samples and use the higher recommended number of PCR cycles.

Table 1. Recommended Number of PCR Cycles for Assessing cDNA Quality by kit type.

| Input Sample | T2 | T10 | T20 | T100 | | High RNA content samples (eg, cell lines) | 10 | 9 | 9 | 8 | | Low RNA content samples (eg, primary cells and nuclei) | 13 | 12 | 11 | 11 |

A small pilot study using 1-4 samples from a new sample type can help guide PCR cycle number selection before scaling up the experiment.

Amplify Library

For Illumina Single Cell 3' RNA Prep workflows, the library amplification PCR cycle number should be adjusted according to the sample type, following the same general principles used for the cDNA quality assessment. It is recommended to follow the cycle numbers in Table 2 for final library amplification. For T10, T20, and T100 kits, consider reducing the cycle number by 1-2 cycles for samples with cDNA QC concentrations > 200 ng/µl.

Table 2. Recommended Number of PCR Cycles for Amplifying Library by kit type.

| Input Sample | T2 | T10 | T20 | T100 | | High RNA content samples (eg, cell lines) | 10 | 10 | 10 | 9 | | Low RNA content samples (eg, primary cells and nuclei) | 16 | 15 | 13 | 13 |

Related Materials:

• Assessing cDNA QC in Illumina Single Cell 3' RNA Prep

• Assessing library QC in Illumina Single Cell 3' RNA Prep

• The importance of pilot studies for Illumina Single Cell RNA Preparation prior to workflow changes

For any feedback or questions regarding this article (Illumina Knowledge Article #9521), contact Illumina Technical Support [e-mail].