10x genomics single cell library prep: low yield of cDNA

[Peris]

I am preparing my 1st 10x genomics single cell library from ~10000 nuclei. After preparing the cDNA I measured its concentration and in qubit it show 0.3 ng/ul. I do understand that nuclei had less RNA content. But does anyone got such low yield of cDNA from ~10000 single nuclei?

Thanks

[nucacidhunter]

I have not seen that low but it depends on starting tissue and cDNA yield should be looked at along the fragment length distribution.

[Peris]

Thanks for your comments. Tissue uses here was mouse cortex. Can it be the reason that the GEM beads captured less than 100 cells? Would you mind if i ask whats the expected concentration of cDNA from ~10,000 single nuclei?

Thanks

[nucacidhunter]

I can comment if you could post cDNA TapeSation or Bioanalyser trace and also the PCR cycles used for cDNA amplification.

If you aimed to capture 10,000 cells and ended up with 100 cells the issue will be steps prior to GEM and cDNA generation.

[Peris]

Here is the image of TapeStation https://ibb.co/M1zV44b. I used 12 cycles for cDNA amplification.

[nucacidhunter]

Profile looks good and can proceed to library prep according to 10x protocol.

[Peris]

Thank you @nucacidhunter. One final query for future references. In your experience within which range I should expect cDNA amount while targeting to recover ~10,000 nuclei; 25-50 ng or >100 ng?

Thanks

[nucacidhunter]

It all depends on the mRNA content of nuclei influenced by many factors. I normally judge by the cDNA profile and look for lack of amplification artefacts and cDNA length distribution rather than quantity.

[Arianna Cerana]

Hello Peris ! Do you have the image of TapeStation of your cDNA?
I’m in the same situation as you and I wanted to see if your cDNA profile was similar to mine.

Thank you in advice!!