The Single Cell Gene Expression Flex Fixed RNA Profiling (FRP) workflow measures RNA levels in fixed samples using probes targeting the whole transcriptome. After whole transcriptome probe panels are added to the sample, each probe pair hybridizes to its target gene and is then ligated. Libraries are generated from the barcoded probes and sequenced.
The final sequencing library structure is similar to Gene Expression solutions for fresh frozen tissue, but the synthetic probe DNA is sequenced rather than the cDNA of a captured transcript. The final library structures for the Gene Expression library and Antibody Capture library (TotalSeq-B or TotalSeq-C) are shown in the appropriate User Guide.
Key advantages of Fixed RNA Profiling include:
- Process formaldehyde fixed or formaldehyde fixed & paraffin embedded (FFPE) samples (single cells or nuclei)
- Increased sample throughput in a single GEM well with multiplexing
- High-sensitivity WTA profiling with efficient sequencing
- Potential to retain multiplet data if each cell has a unique Probe Barcode or Antibody Multiplexing Barcode for Antibody Capture library (labeled "demultiplexible doublet" below). Here are four examples of GEMs with a 10x Genomics gel bead (dark blue) and zero, one or more cells (red or green):
Four examples of GEMs with 10x Genomics gel bead (dark blue) and zero, one, or more cells (red or green).
Cell Ranger v7.0 or later is required to analyze Fixed RNA Profiling data (see library compatibility table).
One or more samples are each uniquely tagged with a Probe Barcode prior to pooling in a single GEM well, resulting in a Gene Expression (GEX) library for each GEM well.
Single- ("singleplex") and multiple-sample ("multiplex") Fixed RNA Profiling Gene Expression workflows are compatible with Cell Surface Protein Feature Barcode technology (hereafter referred to as "Antibody Capture library").
After demultiplexing BCL files to generate FASTQ files, run the cellranger multi pipeline on the FASTQ data to obtain per-sample results.
Fixed RNA Profiling Gene Expression relies on the same underlying Cell Ranger analysis software as Single Cell 3’ Gene Expression solutions, but uses a short read aligner tailored to probe sequences. The probe aligner assigns probe and gene IDs to reads originating from ligation of correctly paired probe halves, distinguishing from potential artifacts and non-probe reads. For fixed samples, Cell Ranger counts oligo ligation events to build the feature-barcode matrix. As a consequence, if the probe hybridizes to a transcript with a variant, that variant will not be present in the sequencing data. Probes are designed to avoid known SNPs. For information on the probe sets, please visit the Probe Set Overview page.
Here are example workflows:
- One sample, one Probe Barcode, one library (CG000691)
- Multiple samples, multiple Probe Barcodes, one library (CG000527)
- Multiple samples, multiple Probe and Antibody Multiplexing Barcodes, two libraries (CG000673)
10x Genomics offers two Chromium products for single-sample gene expression analysis: Single Cell 3' Gene Expression and Single Cell Fixed RNA Profiling for Singleplexed Samples. Key comparisons between these products are summarized below:
| 3' Gene Expression | Singleplex Fixed RNA Profiling (Flex) | |
|---|---|---|
| Feature Barcode compatibility | Antibody Capture (TotalSeq-B), CRISPR Guide Capture, 3' Cell Multiplexing | Antibody Capture (TotalSeq-B or TotalSeq-C) |
| Capture sequence on gel beads | Poly(dT) | Partial capture seq 1 |
| R2 reads from | cDNA made from RNA transcripts | Ligated probes |
| Cell Ranger pipeline | count or multi | multi only |
| R2 reads mapped to | Reference transcriptome | Reference probe set |
| Raw feature-barcode matrix filtered by | Cell calling | Cell calling + probe filtering |
10x Genomics offers two Chromium products that enable pooling multiple samples in the same GEM well: Single Cell 3' Gene Expression with Cell Multiplexing and Single Cell Fixed RNA Profiling for Multiplexed Samples. Key comparisons between these products are summarized below:
| 3' Cell Multiplexing (CellPlex) | Multiplex Fixed RNA Profiling (Flex) | |
|---|---|---|
| Feature Barcode compatibility | Antibody Capture (TotalSeq-B), CRISPR Guide Capture | Antibody Capture (TotalSeq-C) |
| Sample multiplexing tag | Lipid-based CMO tag | Probe Barcode in the probe set, Antibody Multiplexing Barcode for Antibody Capture library |
| Number of libraries | Two (GEX and CMO) or more (GEX, CMO, Antibody Capture, or CRISPR Guide Capture) | One (GEX) or two (GEX and Antibody Capture) |
| Cell Ranger pipeline | multi only | multi only |
| Cell barcode consists of | 10x GEM Barcode | 10x GEM Barcode + Probe Barcode |
| Tag assignment | GEM-level | Read-level |
| Sample assignment by | Reads from singlet cell barcodes assigned to this sample | Reads with a valid barcode (either cell or non-cell assigned to this sample |
| Multiplet detection | Multiplets detected and removed prior to analysis (see true/same-sample doublet diagram above) | Multiplet data is usable if each cell in the GEM has a unique Probe Barcode (see demultiplexible doublet diagram above) |
| Cell calling performed on | The whole GEX library | Individual samples |
| Barcode rank plot | Only one for the whole GEX library | One for each sample in the pool |