We used an enzymatic dissociation procedure (collagenases) in our previous experiments to dissociate tumor tissue for FACS sorting and then RNAseq but it seems that the enzymatic digestion is causing changes in the gene expression.
Can anyone provide me with a good protocol to mechanically dissociate the tumor samples into single cell suspension? I don’t want to use any enzymes. NO ENZYMES
First, best of luck with your sequencing...
While most of the procedures I know require the use of enzymatic digestion (Not sure if Miltenyi kit uses collagenases), please look at the following links for mechanical non-enzymatic dissociation
Article A Simple and Rapid Protocol to Non-enzymatically Dissociate ...
You probably need the dissociator though, and you most likely have it...
More references:
Article Cell dissociation techniques in human breast cancer–Variatio...
Article Technologies for deriving primary tumor cells for use in per...
As very simple as this sounds, you might have to filter the samples a couple of times before flow-sorting...I also hope that mechanical dissociation would be sufficient to give a good yield for the CD45+ or CD45- population that you want...Worst case scenario, if you do not get enough cells, you can use a low-input kit for RNA-seq,..
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