Selecting cycle numbers for High and Low RNA sample types with Illumina Single Cell RNA prep kits
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Assessing cDNA Quality
Illumina Single Cell 3’ RNA Prep kits require the 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 users do not know if their sample type is High or Low RNA content it is best to adhere to the general recommendation in the reference guides for assessing cDNA quality PCR cycle number as seen in Table 1.
Cultured cell lines should be treated as High RNA sample types and use the lower number of recommended cycles.
Primary cells isolated from patients or animals as well as nuclei from all sample types should be considered Low RNA sample types and use the higher number of recommended PCR cycles.
Table 1: Recommended Number of PCR Cycles for Assessing cDNA Quality
Running a small pilot study of 1-4 samples with a new sample type can inform PCR cycle number when scaling up or increasing number of samples in an experiment.
Amplify Library
Illumina Single Cell users must adjust their amplify library PCR cycle number according to their sample input following the same general rules as with Assessing cDNA Quality. It is recommended to follow the cycle numbers in Table 2 for amplifying the final library. For T10, T20, and T100 kits, decrease 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
For additional information, search the following Illumina Knowledge articles:
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
Related Materials:
For any feedback or questions regarding this article (Illumina Knowledge Article #9521), contact Illumina Technical Support techsupport@illumina.com.
