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!
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!
Comment