Hi all,
our collaborators would like to perform an RNAseq experiment from a 2-cell co-culture system.
For this experiment they would like to co-incubate fibroblasts and macrophages from 2 different backgrounds (Strains) of the same organism.
After cultivating the cells, the bulk analysis should be able to separate both cell types. Either doing the analysis separately for each cell type, or doing it together, while being able to distinguish the two cell types.
As this is also new to me, I was wondering, if anyone has heard of such analysis and whether or not this is feasible.
Would it possible to have the RNA samples, that contain both RNA from fibroblasts and RNA from macrophages to sort the reads to the corresponding cell type and analyze them separately?
The idea behind this kind of analysis is that the SNP should be different enough to be detected. One cell would have a SNP pattern from the first strain, while another one should have the SNP pattern of the second strain. They should have both different, but also overlapping transcriptome
In other words, this method would only separate mRNAs (i) with a SNP(s), (ii) with a differential transcript pattern, and (iii) with information on cell-specific differences. Example: A macrophage-specific scavenger receptor from the first strain would not be expressed on the other cell in the pool and would have (in theory) a different SNP.
Does anyone know of this kind of analysis? Any papers about it or any other sources?
Thanks in advance
Assa
Hi Assa,
Wouldn't it be best to sort the cells anyway? They will have very different surface antigen profiles that should make this straightforward and give you confidence that the right cells are in the right wells. You can then also exclude dead cells, doublets and debris from the intact cells and if necessary see the differences between (pre-)apoptotic and differentiating and quiescent healthy cells. The means to achieve this are all nicely dealt with a paper from EMBL - Ordoñez-Rueda et al. Cytometry Part A, 2020. This can all be achieved with a few simple probes - DRAQ5, DAPI and AnnexinV and a discriminatory antibody marker (or two) for fibroblasts vs macrophages.
If you want to go forward with isolated nuclei then Preissl (now at UCSD) strongly advocates a DNA marker for the FACS sort deposition of these events to exclude debris and typically uses the far-red DNA probe DRAQ7 for this. He gave a nnice webinar on this recently (available at Selectscience, sponsored by Sony).
I hope this helps!
Regards,
Roy
Hi Assa,
assigning reads from bulk measurements to different cell types that were present when you harvested the RNA will be hard. It is certainly possible to assign the reads that contain particular SNPs, but that means you will be left with a massive number of reads that you cannot assign (you would need to have several differentiating SNPs per gene). On the analysis side, I would be very, very careful to make strong claims about cell type specificity.
As Roy Edward pointed out, you might want to try FACS to differentiate between the cell types, which would be way preferable. If you don't have access to FACS, you might try and enrich your cultures for either one or the other cell type to get a feeling of what the typical gene expression profiles might look like.
Best,
Lukas
Hello Assa
A single-cell RNAseq would be an option. you will have separate results based on your cell types. Even you might have 20 different cell types still you will be able to have the proper analysis specifically filtered by cell types. The price is much more than a standard RNAseq and the analysis pipeline needs a higher spec computer. You better use cloud computing.
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