From Fresh to Fixed: Matching Your Sample Type to the Right 10x Single-Cell Prep

Single-cell RNA-sequencing can be a game-changer when evaluating complex samples. This method allows us to capture the heterogeneity in a sample by allowing full transcriptome evaluation of each individual cell. While historically we only considered cryopreserved cells for 10x studies, a wide variety of sample types can now be used.

The key is to match your starting material with the optimal prep method.

From fresh and cryopreserved cells to frozen tissue, FFPE samples and blood, your sample type plays an important role in choosing the right 10x workflow for your study, whether you’re working with GENEWIZ or in your own lab.

Start With Your Sample

Every 10x single-cell protocol ultimately needs a suspension of viable or preserved single cells or nuclei. Minimizing cell aggregates, dead cells, ambient (free-floating) RNA, and reverse-transcription inhibitors is critical to getting clean data. The accepted sample types generally fall into these buckets:

Fresh cells and freshly dissociated tissue

This sample type is great for direct loading with 10x. You must ensure viability is >80% with minimal debris, and a cell size under ~30 µm to avoid clogging the microfluidics.

Cryopreserved cells / snap-frozen tissue for dissociation

Because cryopreservation can lower viability, test post-thaw viability to ensure it is >80%. Dead-cell removal can improve viability, although below ~50% risks ambient RNA contamination and is typically not loaded.

Snap-frozen tissue can be dissociated and used as a single-cell suspension.

Methanol-fixed cells

This sample type is ideal for fragile cells that are not viable post-cryopreservation.

Formaldehyde (PFA) fixed cells, nuclei, and tissue

PFA-fixed cells preserve the biological snapshot and allow storage at −80°C for up to ~12 months. This is best for samples that are not ideal for cryopreservation.

Formalin-fixed, paraffin-embedded (FFPE) tissue blocks and sections

FFPE is for archival human or mouse samples only

Isolated nuclei

This starting material is best used when intact viable cells or specific cell types are hard to recover from frozen tissue. Nuclei reduce dissociation artifacts and cell-size capture bias, though expect ~20–40% fewer genes and ~20–60% fewer UMIs than whole cells, depending on the sample type.

Fixed whole blood

For human or mouse samples only, direct fixation of whole blood preserves fragile cell types and simplifies handling.

Choose the Right 10X Workflow

10x uses three main methods for RNA-sequencing, which have variable input sample types and potential add-ons during sample processing.

Universal 3′ Gene Expression

The “standard” method uses poly(dT) capture of the mRNA 3′ end, making it species-agnostic across all animals. Additionally, this method is compatible with cell surface protein, CRISPR screening, and cell multiplexing add-ons.

Universal 5′ Gene Expression

This method captures the 5′ end via a template-switch reaction, which uniquely enables paired, full-length V(D)J TCR/BCR immune repertoire profiling for human and mouse samples. If immune repertoire profiling is a key goal of your study, 5' Gene Expression is the workflow to consider.

Flex/Apex Fixed RNA Profiling

Flex/Apex is a whole transcriptome probe-based method for human or mouse samples only. It is ideal for degraded RNA, including FFPE, PFA-fixed material and blood from PAXgene tubes.

Match Your Sample to Your Workflow

A Simple Rule: Let Your Sample Lead the Way

The most important decision in a 10x single-cell experiment happens before any cell touches the instrument: let your sample type steer your chemistry.

Working with fresh or cryopreserved samples and need maximum flexibility across species?

Choose 3′ or 5′ Gene Expression.

Need immune repertoire?

5’ Gene Expression is for you.

Working with precious fixed or FFPE archival samples?

Go with Flex/Apex.

Planning a single-cell study? Our experts can consult with you on your project, from sample preparation and workflow selection to study design. Sign up for a complimentary consultation with GENEWIZ today.