Question: What is the expected size and concentration of Flex Gene Expression Libraries?
Answer: After library construction, Flex Gene Expression libraries are run on the BioAnalyzer, Tapestation, or LabChip at a 1:80 dilution for qualitative analysis. The traces should resemble the overall shape of the electropherograms shown in the User Guide. We expect a good Flex Gene Expression library trace will appear as a single discrete peak with an average library size of ~260 bp.
Representative Flex Gene Expression Library Trace:
Flex Gene Expression libraries should ideally fall within the target concentration range of 50 - 200 nM. However, if the concentration is between 10-50 nM or between 200-500 nM and if the libraries do not contain low or high molecular weight peaks, sequencing can still be performed.
Representative Flex Gene Expression Library Trace for Under-cycling:
Higher proportion of low molecular weight products present in the library
Representative Flex Gene Expression Library Trace for Over-cycling
Additional higher molecular weight peaks present in the library trace
Recommendations for Under- or Over-cycling:
Sample Index PCR can be repeated with a greater or fewer number of PCR cycles, respectively, using the leftover pre-amplified product from the first PCR. Additional Amp Mix can be obtained from the Fixed RNA Feature Barcode Kit (PN-1000419).
If you've generated unexpected library traces, please reach out to 10x Genomics Technical Support (support@10xgenomics.com) for guidance.
Product: Flex Gene Expression