Been meaning to post this for a while. Anyway, in case you're interested, here is a comparison of mean number of RNA per cell measured by RNA FISH to FPKM as measured by RNA-seq for 26 genes (bulk and also combined single cell RNA-seq). Experimental details in Olivia's paper. We used a standard RNA-seq library prep kit from NEB for the bulk, and used the Fluidigm C1 for the single cell RNA-seq. Cells are primary human foreskin fibroblasts.
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| Bulk RNA-seq vs. RNA FISH (avg. # molecules per cell) |
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| Single cell RNA-seq vs. RNA FISH (avg. # molecules per cell), linear scale |
Probably could be better with UMIs and so forth, but anyway, for whatever it's worth.
It would be interesting to see in addition to this also a plot where on y-axis one would have number of reads which contain (or map) the FISH probes sequences (which the one used in the FISH experiment) and on x-axis the FISH counts.
ReplyDeleteI guess that this would correlate much much better with FISH than RPKM (or CPM or TPM) because the coverage of sequencing is not uniform at all.
I'm not so sure. I think that RNA FISH as a whole is a proxy for the number of RNA molecules, as is RPKM (or CPM or TPM or whatever). Where the probes bind shouldn't influence our number of RNA molecules (we have shown this), so I don't think it should really correlate more or less with RPKM if we just focus on those areas of the transcript for counting RNA-seq reads. But I could be thinking about this wrong.
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