Hello All,
I am going to conduct an RNA-Seq experiments on In-vitro differentiated neurons from the mESCs. At the end of the differentiation, I usually get a lot of cell death in the cultures and I am concerned that these dead cells can interfere with the RNA-Seq analysis. I want to know if people care about removing the dead cells before extracting RNA for the sequencing or what is the general norm in the field.
Any suggestions/opinions/tips?
Thank you all!
Best,
Sandeep
In our lab we do RNA-seq on mouse ES cells. During differentiation a large proportion of cells will die and float around in the medium. To prevent as much as possible the RNA reads of dead cells I wash the cells extensively with PBS (2-3x) before harvesting with TRIzol. This should be enough to get rid of most of the dead cells. Any leftover dead cells will probably be not more than 1% of the total cell population and will not matter that much in RNA-seq.
Virginia Tech, S. Turner - we have developed a novel product to separate out dead cells from live with >90% effectiveness, for sample prep to improve live single cell RNA sequencing data quality, and are looking for core sequencing laboratories that might like to collaborate in validating the application. Contact [email protected]