Question: What is the expected size and concentration of Fixed RNA Profiling Libraries?
Answer: After library construction, Fixed RNA Profiling 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 Fixed RNA Profiling library trace will appear as a single discrete peak with an average library size of ~260 bp.
Representative Fixed RNA Profiling Library Trace:
Fixed RNA Profiling 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 Fixed RNA Profiling Library Trace for Under Cycling:
Higher proportion of low molecular weight products present in the library
Representative Fixed RNA Profiling 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 Library Amplification Kit (PN-1000249). The recommended number of cycles are listed below:
If you've generated unexpected library traces, please reach out to 10x Genomics Technical Support (firstname.lastname@example.org) for guidance.
Product: Fixed RNA Profiling, Single Cell Gene Expression Flex