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SeqReady guide · Single-cell

Single-cell RNA-seq sample prep: viability, cell counts, and cells or nuclei

One single-cell library can cost over $3,000, and you only get one go with each suspension. The good news: many problems are visible under the microscope before you load.

By Eric A., MSc Molecular BiologyNovember 20264 min read
After 4 minutes you'll know

Single-cell days are stressful: expensive reagents, a booked slot, and cells that don't like waiting. Most failures start at the bench, hours before the instrument. Here's how to walk in calm.

01Where things go wrong

⏱ the clock is running: cells are stressed from here on1Tissue2Dissociate3Filter + count4Core QC5Loaddamage startscounts slip
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From the moment you dissociate, every minute counts. Damage and bad counts are the two usual culprits.

02What the core sees under the microscope

Ready to loadToo many dead cellsClumps and debris
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Illustration. Cores check viability (live vs dead stain), concentration, clumps and debris. Ask for their exact limits early.

03Why dead cells matter so much

Dead and dying cells leak their RNA into the buffer. That free RNA gets captured with every real cell, adding a background of genes that don't belong. So a few dead cells don't just waste themselves; they blur the whole run.

~90%0%50%100%talk to the core before the daywhere many aim
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Viability targets depend on the core and platform. Many guidelines aim around 90%. If your tissue can't reach it, tell the core before the day.
Watch out
Low viability is hard to fix afterwards. Tune your dissociation on spare tissue first.

04Counting you can trust

  1. Filter, then countRun the suspension through a cell strainer just before counting to remove clumps.
  2. Use a live/dead stainTrypan blue or a fluorescent stain, so you count live cells, not debris.
  3. Count twiceIf the two counts differ a lot, mix gently and count again. A wrong count means too few cells recovered, or extra doublets.

05Cells or nuclei?

🔵 Whole cells

  • Fresh tissue or blood that dissociates well
  • You need surface proteins (CITE-seq)
  • You want the most RNA per cell

⚪ Nuclei

  • You only have frozen tissue
  • Large or fragile cells (neurons, fat, muscle)
  • Warm digestion would change expression
  • You want chromatin data too (multiome)

Nuclei data look a little different (more intronic reads). That's normal, and pipelines have settings for it.

06Plan the day backwards

Book the core slot firstThen plan dissociation backwards from it.
Keep cells cold, move quicklyUnless your protocol says otherwise.
Bring a spare aliquotIn case the first count is off.
Write down timingsIt's the first thing a core asks if something goes wrong.

A good single-cell run is mostly decided before you reach the instrument. Healthy cells, an honest count, and a plan, and you can walk into the core with confidence.

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Common questions

What viability is "good enough"?

It depends on the core and platform, so ask. Many guidelines aim high (around 90%) and flag samples well below that. If your tissue can't reach it, talk to the core before the day, not after.

Can I fix my samples and run them later?

Some platforms and kits accept fixed cells, which takes the time pressure off the day. Check what your core supports before you choose this route.

Why did I get fewer cells than I loaded?

Every platform recovers only part of the cells loaded, and a wrong count makes it worse. Ask your core what recovery to expect.

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About the author
Eric A.

MSc in Molecular Biology from the University of Debrecen, Hungary, with research on nanopore sequencing for hospital infection control. He has also worked on the provider side of NGS services, helping universities and hospitals scope their sequencing projects.

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