Hi,

I'm, analyzing a single-nuclei dataset with not-so good quality, and I am trying to understand what went wrong.

These are cancer samples. Biopsies of metastases found in the liver of 3 patients. Pre-/post-treatment.

I ran velocyto to get the % intronic reads / total reads on the raw barcode matrix.

I'm assuming that droplets containing mostly ambient RNA coming from the cytoplasm of broken cells should have low %intronic. However, my "empty" droplets range 30%~60% intronic reads.

Which values of %intronic should I expect in cancer cells and in surrounding "healthy" liver tissue from cytoplasmic or intronic origin? I assume that, ambient RNA coming from the (unwashed) broken cytoplasm of otherwise "successfully captured" nuclei should lie at 0% intronic reads, no? What would the % approximately look like if it came from "whole broken cells" (cytoplasm + nucleus) ?

It looks to me like the ambient washout protocols were successful, and what I'm seeing on the empty droplets is... mostly ambient RNA coming from broken nuclei? Maybe because of harsh FACS-sorting?

Any ideas of what I'm seeing here?