Hi this is my first post so please forgive me if I posted this at the wrong place.
So the goal is to perform single-cell RNA-seq (everybody seems to be doing that nowadays).
I optimized a protocol to successfully dissociate my study organism (a marine invertebrate), and got clean cell suspension with no obvious debris.
Now I'm trying to take it one step further by fixing the cells with ACME solution (10.1186/s13059-021-02302-5), so I can save experiments and do scRNAseq later.
After thawing and resuspension in 1x PBS/0.04% BSA, I saw large debris (see picture) that weren't there before freezing. I reason that since the cell counts were lower than before fixation, it's probably from cells that died from the fixation and freezing process.
my problem is, I've tried lowering centrifuge speed/time but still didn't seem to get rid of most the debris.
I'd appreciate people's advice on how to proceed: either 1) the debris will not cause trouble with 10X single-cell sequencing, or 2) ways to reduce the debris.
PS. I do have access to a FACS machine which I could potentially use to select/enrich the cells, but I wanted to see if people have simpler methods I could try before I jump on the FACS machine.
Thanks a bunch!
This forum focuses on bioinformatics. You should post this at SeqAnswers.com or biology stackexchange.
The first one is very old, the second one an utter mess imho, I recommend asking this at Reddit, there are multiple subReddits focused on NGS, molecular biology etc.