Single cell (10X)

10X Genomics Next GEM technology

GTF operates 10X Genomics solutions for high throughput single cell profiling.

10X Genomics trick is based on single cell/nucleus emulsion. A DNA barcode, unique in each droplet, is linked to DNA/cDNA. The emulsion is broken and the subsequent sequencing library generation steps are performed bulk.

Up to 10’000 cells/nuclei can be assayed in a single well, i.e. up to 80’000 cells per chip. However, the more cells or nuclei are loaded, the more multiplets may be present (see for instance this 10X Genomics note). In addition, more cells mean higher sequencing effort required (see below). A common target is in the range of 5’000 cells/nuclei per well.

Applications

Main 10X Genomics single cell applications are:

  • Single cell transcriptomics
  • Single cell epigenomics (chromatin) with ATAC-Seq
  • Combine transcriptomics and ATAC-Seq (” multiome”)

Single cell transcriptomics is not restricted to RNA profiling since each single cell analyzed can optionally be further characterized with:

  • Immune repertoire profiling (Human or Mouse B or T cells V(D)J sequencing)
  • CRISPR library sequencing for Perturb-Seq (also known as CRISP-Seq or CROP-Seq)
  • Cell surface protein expression with DNA barcoded antibodies (also known as CITE-Seq or TotalSeq)
  • Antigen Specificity with dCODE Dextramer

Schedule an experiment

Samples should be processed as quickly as possible after the cell sorting. Accordingly, experiments must be carefully scheduled with our staff. Please contact GTF as early as possible when designing your project.

In order to maximize cell viability, the 10X Chromium may be operated in one of the Lausanne University Cell Sorting facility (FCF, at Agora or Biopole) laboratory by a specialized GTF technician (service available upon request).

Cells or nuclei preparation

Cells or nuclei preparation is of user’s responsibility !

The cells/nuclei suspension quality is crucial for a successful single cell project. Cells/Nuclei must be perfectly dissociated, their density accurately quantified, and viability must be above 70% (for transcriptomics applications). Nuclei preparation is even more challenging since viabilitycheck does not apply (see 10X note).

Any manipulation (e.g. concentration by centrifugation) or waiting time can impact cells/nuclei quantity and quality. In particular, cell count and viability output metrics from cell sorters cannot be taken for granted. It is more than highly recommended that a final quality control step is performed on the very final material you provide us. You may want to use our Moxi V cell counter which is perfect for 10X Genomics application (see this technote).

Failure to provide good quality starting material will inevitably result in suboptimal, sometimes useless, results. Data quality can only be assessed after sequencing, meaning that expensive 10X and Illumina reagents are engaged, whatever the outcome is.

In any case, we strongly recommend that pilot experiments are performed in order to validate the cell preparation procedures.

The Chromium loading efficiency is in the range of 40% to 60%. This means that only ≃ 50% of the cells/nuclei loaded in the chip’s wells will actually be assayed. Given there is also generally around 50% yield for the cell preparation step (cell sorting and subsequent washings), we recommend starting the preparation procedure with at least 4-fold the amount of targeted cells/nuclei.

As a starting point, please refer to the following 10X genomics documents: Sample Preparation Tips for Single Cell Gene Expression (transcriptomics applications), Nuclei Isolation for Single Cell ATAC Sequencing (ATAC-Seq application), or Nuclei Isolation for Multiome (Gene Expression + ATAC seq from the same nuclei).

Cell or Nuclei Multiplexing

Samples may be multiplexed before being loading on the 10X Chromium chip.

The process is based on an extra DNA label added to the cells or nuclei.
This saves reagents in case many samples have to be run in parallel, but decrease the number of individual cells assayed for each condition.

10X Genomics proposes an official solution with the CellPlex technology. So far, it is supported with the 3′-gene expression kits only.
Multiplexing can also be achieved with DNA barcoded antibodies as a process called Cell Hashing

Setting up a multiplexing experiment is not trivial. Please contact GTF for further information.

Spatial Transcriptomics

10X genomics offers Spatial Transcriptomics solutions.

It is available for fresh frozen samples, as well as FFPE material (but for human and mouse only).
Be aware the current resolution is pretty low, with 55 um wide spots.

Spatial transcriptomics involves multiple fields of expertise, not related to genomics, upstream of the sequencing library preparation step. Experimental procedures must be discussed in details with GTF, before planing any experiment.

Digital Spatial Profiling technology (DSP also known as GeoMx) from Nanostring is also available, but not operated by GTF. Please inquire for further information.

Library preparation

Once the user has prepared the cells/nuclei suspension, GTF will operate the 10X Genomics Chromium and generate the sequencing library. All 10X genomics reagents as well as advanced library quality control are included.

Chromium cell processing can optionally be split into several batches (i.e. chips) in order to avoid samples waiting too long. This must be discussed ahead with GTF.

We perform 10X library sequencing on the NovaSeq. The output is 400M filtered/usable reads per lane. The total amount of sequencing required is directly dependent on the number of cells loaded.

For ATACseq, the 10X Genomics recommendation is 25’000 reads per cell (Human or Mouse genomes).

For RNAseq, 10X genomics recommends around 25’000 – 50’000 reads per cells but this number is greatly influenced by the complexity of the transcriptome (see figure below as well as this Technical Note). In addition, special interest in low expressed genes (e.g. transcription factors or long non coding RNA) may impose a higher sequencing depth.

A good practice is to request a single sequencing lane to start with. Further sequencing can then be requested after quality control for validating cell number, evaluating transcription saturation, and inspecting the presence of some genes of interest.

Single cell transcriptomics sequencing saturation

Sequencing

For Spatial transcriptomics the recommendation is 50’000 reads (fresh frozen material) or 25’000 reads (FFPE material) per spot. A capture area is made of 5’000 spots but only partially covered by the tissue slice. Sequencing depth requirement can be calculated by estimating the percentage of capture area occupancy.

Data analysis

GTF 10X Genomics service includes primary analysis with Cell Ranger software using standard settings. Deliverables are fastq files as well as the corresponding Cell Ranger count file outputs.

Secondary analysis, with refined clustering or pathway analysis for instance, is not proposed by GTF.

Coming soon

A newer single cell processor, the Chromium X, will soon be available at GTF.

It will allow runing the 10X High Throughput kits, reducing the processing cost for large number of cells, especially when using multiplexinhg strategies.

The Chromium X will be key for the newer Single cell fixed RNA profiling application from 10X genomics (human and mouse only).
This is a revolution for single cell transcriptome profiling since it will simplify the processing of fragile cells, will simplify time course analysis, and when combined with multiplexing, will maximize the number of cells processed at once (up to 128’000 cells per well).