Single-cell transcriptome, small library size

[lmw.bio]

Recent preparation of single-cell transcriptomes using Smart-Seq v4 Ultra Low Input RNA Kit generated smaller size pool than usual. Concentrations are o.k. according to the Qubit and I have been able to amplify various protein coding genes with specific primers, however the size distribution of the full library is smaller than expected. The cDNA doesn't seem to be degrading (at present) but perhaps it degraded before purification with AMPure beads? Or perhaps the Smart-Seq V4 reaction didn't work properly?

I'm looking for answers as to 1) WHY this happened and 2) HOW to salvage the sequences that I do have that seem to be of good quality.

Attached (WTAs_bioanalyzer.pdf) are results from the Bioanalyzer. Sample 1 is an old and typical single-cell transcriptome and samples 2, 3 and 4 are the failed transcriptomes in question.

Thanks for your input!

[nucacidhunter]

I wonder if the profiles are from final library or amplified cDNA and if input was single cell or purified RNA. If they are final library what prep method was used.

[lmw.bio]

Oh, thank you, I wasn't very clear. The input for the Smart-Seq kit was single-cells. The profiles I shared are of the total output from the Smart-Seq reaction, therefore they are individual single-cell amplified transcriptomes (after bead cleaning) of the double-stranded cDNA (before indexing or other library prep).

[luc]

Sorry, I have not seen this pattern before.
I would suggest to finish the library prep for one of these and then generate a small amount of sequence data to continue the troubleshooting based on these data.

[nucacidhunter]

It could be due to using unvalidated media or RNase contamination of plastic ware or reagents. If you have followed the kit protocol, Clontech/Takara tech support should be able to help troubleshoot. I do not think that proceeding with the cDNA will give useful publishable data.