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Cell Ranger


10x Genomics
Chromium Single Cell Gene Expression

What is Fixed RNA Profiling?

The Single Cell Fixed RNA Profiling (FRP) workflow measures RNA levels in samples (single cells or nuclei) fixed with formaldehyde, 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.

Key advantages of Fixed RNA Profiling include:

Four examples of GEMs with 10x Genomics gel bead (dark blue) and zero, one, or more cells (red or green).

Table of Contents

Analyzing Fixed RNA Profiling data with Cell Ranger

Cell Ranger v7.0 or later is required to analyze Fixed RNA Profiling data. Cell Ranger v7.1 supports analysis of single cell FFPE (scFFPE) datasets.

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. A single-sample ("singleplex") Fixed RNA Profiling experiment with one Probe Barcode and one sample is compatible with Cell Surface Protein Feature Barcode technology, and can generate a paired GEX and Feature Barcode library (hereafter referred to as "Antibody Capture library"). Here are two example workflows:

One sample, one Probe Barcode, two libraries: singleplex FRP

Multiple samples, multiple Probe Barcodes, one library: multiplex FRP

After demultiplexing the BCL files with cellranger mkfastq, run the cellranger multi pipeline on the FASTQ data to obtain separate per-sample output files. 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. The probe design avoids known SNPs. For information on the probe sets, please visit the Probe Set Overview.

Comparing 10x Genomics single-sample solutions

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
Feature Barcode compatibility Antibody Capture, CRISPR Guide Capture, 3' Cell Multiplexing Antibody Capture (TotalSeq-B)
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
R2 reads mapped to Reference transcriptome Reference probe set
Raw feature-barcode matrix filtered by Cell calling Cell calling + probe filtering

Comparing 10x Genomics multiplexing solutions

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 Multiplex Fixed RNA Profiling
Feature Barcode compatibility Antibody Capture, CRISPR Guide Capture Not compatible
Sample multiplexing tag Lipid-based CMO tag Probe Barcode in the probe set
Number of libraries Two (GEX and CMO) One (GEX)
Cell Ranger pipeline multi multi
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

Next steps