Single-cell Flex v2

Single-cell gene expression sequencing with 10x Genomics Flex v2 took about half of my placement at Cincinnati Children's Research Foundation. I prepared the protocol, the samples and the custom probes, ran 88 samples through the full protocol for a mouse study, and analyzed the sequencing data with Cell Ranger.

Where
Cincinnati Children's Research Foundation, Research Student II, August 2025 to May 2026
Sequencing
Illumina machines
Analysis
Cell Ranger
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Cincinnati Children's Research Foundation · Aug 2025 to May 2026

Single-cell Flex v2

About half of my placement was single-cell gene expression with 10x Genomics Flex v2.

I prepared
the protocol, the samples and the custom probes
I ran
88 samples through the full workflow, for a mouse study
I analysed
the data with Cell Ranger. Sequencing used Illumina machines.

Follow one run, stage by stage My account; no lab log

Stage 1 of 12

88 samples

88 samplesEighty-eight sample cards in a grid of eight rows and eleven columns, a mouse outline, and four labels: protocol, samples, custom probes and Cell Ranger. One card links to a cell. 88 sample cards Mouse study Protocol Samples Probes Cell Ranger One card holdsmany cells

I ran 88 samples through the full Flex v2 workflow for a mouse study.

  • One sample card

One sample holds many cells.

Stage 2 of 12

Fix the sample

Fix the sampleA pipette adds fixative to a tube of cells. A centrifuge spins the tube and leaves a pellet. A magnified cell changes from a thin dashed edge to a thick solid edge while its RNA stays inside. Cells ornuclei Fixative Spin Pellet Fixed cell,RNA held in place

Fixation preserves the sample for later RNA measurement with probes.

  • Fixed cell
  • RNA

Tissue starts with a separate fixation and dissociation route.

Stage 3 of 12

Hybridise the probes

Hybridise the probesTwo probe tubes and a plate feed a magnified fixed cell. A left probe half and a right probe half bind side by side on an RNA strand with a small unjoined gap. A custom pair binds the same way. Mousepanel + Customspike-in Inside one fixed cell 3′5′ LHSRHS Custom pair Target RNA

Probe pairs bind neighbouring RNA sequences. Custom probes join the whole transcriptome panel.

  • LHS probe
  • RHS probe
  • RNA
  • Custom pair

A gap between the two halves means they are not joined yet.

Stage 4 of 12

Barcode the samples

Barcode the samplesA barcode oligo plate, a pipette and a thermal cycler sit above two samples. For each sample a barcode oligo with a violet sample barcode binds the handle on the right probe half. The joints stay open. Barcodeoligo plate Hybridise Sample A Samplebarcode Sample B Samplebarcode

Separate barcode oligos label samples before pooling.

  • Sample barcode
  • Probe handle

Sample barcode, GEM barcode and UMI are three different tags.

Stage 5 of 12

Wash and pool

Wash and poolThree sample tubes go through a centrifuge. Unbound probe pieces leave with the liquid. Washed cells flow into one pool tube, where cells carry different sample marks. Centrifuge A B C Pool Unbound probes Mixed samples

Wash away unbound material, then pool the labelled samples.

  • Sample marks
  • Probe halves

Stage 6 of 12

Make GEMs

Make GEMsA chip is loaded with pooled cells, gel beads and oil, then slides into a Chromium instrument. In a schematic channel, oil pinches the stream into droplets. Most droplets hold a bead and a cell, one is empty. Chromiuminstrument Cells Beads Oil GEM Two samplescan sharea GEM

The Chromium instrument partitions pooled cells with barcoded gel beads into GEMs.

  • Gel bead
  • Cells
  • Oil

GEM means gel bead in emulsion. Some GEMs are empty.

Stage 7 of 12

Ligate in the GEM

Ligate in the GEMStrip tubes enter a thermal cycler. In an enlarged GEM the gel bead dissolves and releases primers, the cell opens, and the gaps between probe halves and the barcode oligo close. Thermal cycler ◆○+ ◆Bead dissolves○Cell opens+Gaps close

Inside GEMs, ligation joins probe pairs and barcode oligos.

  • LHS probe
  • RHS probe
  • Joined

Ligation closes each gap while the probes sit on the RNA.

Stage 8 of 12

Add GEM barcode and UMI

Add GEM barcode and UMIBead primers share one blue GEM barcode and carry different amber UMIs. One primer lands on the capture sequence and a polymerase extends it along the ligated probe product. Brackets name each part. Bead primers Same GEM barcode,varying UMIs Pol ProbeinsertSamplebarcodeGEMbarcodeUMI

Released bead primers add the GEM barcode and UMI by extension.

  • GEM barcode
  • UMI
  • Sample barcode

UMI means unique molecular identifier.

Stage 9 of 12

Recover and amplify

Recover and amplifyRecovery agent breaks the emulsion and the tube separates into an aqueous layer and an oil layer. Oil goes to waste. PCR copies the products and keeps their barcodes. Magnetic beads clean up the DNA. Break GEMs Pre-amplify Clean up Recoveryagent Aqueousphase Waste Copies keepthe barcodes Beads onmagnet Clean DNA

Break the emulsion, recover the DNA products, copy them by PCR and clean up with magnetic beads.

  • Cleanup bead
  • Aqueous phase

Cleanup beads are not the gel beads from the GEMs.

Stage 10 of 12

Build and check the library

Build and check the libraryA library molecule builds up segment by segment with adapters, indexes, the probe insert, sample barcode, GEM barcode and UMI. A size trace and quantification curves follow. Sample Index PCR i5indexProbe insert withbarcodes and UMIi7index Check sizeMeasure amount

Sample Index PCR adds adapters and indexes. Size and concentration are checked before sequencing.

  • Library index
  • Sample barcode

Library indexes are separate from the sample barcode.

Stage 11 of 12

Sequence

SequenceLibrary molecules enter a flow cell and bind to oligos on its surface. Copies build a cluster. A cycle of add base, image and unblock repeats. Base calls become BCL files, then FASTQ files. Flow cell Template Cluster Add base Image Unblock Repeat for each base BCL Demultiplex FASTQ

Libraries bind a flow cell, form clusters, and are read by sequencing by synthesis.

  • Library strand
  • New base

Stage 12 of 12

Count with Cell Ranger

Count with Cell RangerA terminal shows a generic cellranger multi command with FASTQ, config and probe set files. Repeated reads collapse to one count. A matrix fills with features as rows and cell barcodes as columns. Output files follow. $ cellranger multi \ --id=flex_run \ --csv=config.csv FASTQConfigProbes Cell barcodes Features Copies count once MatrixQCReport Web summary

Cell Ranger assigns reads and counts distinct probe ligation events into a gene-by-cell matrix.

  • Input and output files
  • filtered_feature_bc_matrix/
  • web_summary.html
  • metrics_summary.csv

The command is a generic example.

Illustrative Flex v2 workflow.

What I did

I prepared the protocol, samples and custom probes, and analysed the sequencing data with Cell Ranger.

Sources

Sources

I drew these diagrams using the linked 10x Genomics and Illumina workflow descriptions.

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