Question: troubleshooting help: scRNA-seq cDNA are far longer than expected (~6K bp) after reverse transcription
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3.5 years ago by
maxwellb0
maxwellb0 wrote:

As the title says, my lab mate and I are generating gene libraries for a single cell RNA-seq experiment. We're following SMRT-seq 2 protocol for reverse transcription. Our yield is within the optimal range after 20 PCR cycles (300pg/uL). Post-amplification fragment length distribution on the tape station (or bioanalyzer) should show a peak at ~2k bp. We're seeing a relatively broad distribution, with a peak at ~6k bp. I've attached an image of our gel with two representative lanes showing fragment distributions.

I've searched for troubleshooting advice on how to interpret such a result but can't find anything online. Has anyone had this problem? How did you interpret it? How did you fix it?

Tape Station Run

rna-seq gene library • 1.7k views
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