Single cell initiative

Depending on the technology, single cell analyses require different input material. Apart from spatial approaches, which can be applied on fresh frozen or formalin-fixed paraffin embedded (FFPE) tissues, most single cell technologies necessitate dissociated cells or nuclei. These can be obtained from cultured cells, blood cells or tissues.

A good cell suspension quality is key for a successful single cell experiment. Cell suspensions are usually obtained by enzymatic digestion and/or mechanical dissociation. Take your time to optimize your protocol, in order to reduce cell suspension handling time, debris, aggregates and/or cell death.

Once a good cell suspension has been obtained, single cells/nuclei need to be isolated. For this, several options exist:

10X Chromium 

Available at the NGS platform, as well as at the Cytometry facilities of Orsay and St Cloud/Suresnes, the 10X Chromium X uses advanced microfluidics to perform single cell partitioning and subsequent barcoding. It is an integrated part of all 10X single cell applications.

Flow cytometry

Available at the Cytometry platform, cell sorters allows sorting your cells according to the presence of up to 50 markers (e.g. cell surface receptors) and thus to sort only the cells of interest for the subsequent single cell experiment.

CellenONE

Available at the Custom Single Cell Omics platform, the CellenONE is an image-based single cell sorting device that is ideal for low amounts of cells (<1000). The recovery rate is very high and dead volumes are small.

Single cell DNA and RNA sequencing allows you to study the genome and the transcriptome of individual cells. Different methods exist, which are complementary. The method of choice will depend on your biological question and your input material:

3’ scRNA sequencing

3’ RNA sequencing allows to quantify gene expression and to identify cell populations. In 3’ scRNA-Sequencing, poly(A)+ mRNAs are reverse transcribed using a polydT primer. This polydT sequence is located directly adjacent to the barcodes required for the identification of unique cells and for sequencing. The generated cDNA molecules are amplified and then fractionated. Only the 3’ ends of the transcripts bear the barcodes, are represented in the final libraries and will be sequenced. 3’ scRNA-seq represents a relatively easy way to quantify the poly(A)+ mRNAs of single cells.

3’ scRNA-seq at the NGS platform

3’ scRNA-seq is available in routine at the NGS platform using the 10X Genomics pipeline. The required input material is 1,000 to 20,000 cells or nuclei and the recovery rate (yield) is around 50% of loaded cells/nuclei. The sequencing recommendations are around 50-100k reads per cell or nucleus.

Using feature barcodes, 3’ scRNA-seq can be combined with cell surface proteins expression using specific antibodies at the single cell level (CITEseq) or to increase the multiplexing capacity (Cell Hashing). Effects of CRISPR perturbations can also be assessed at single cell resolution, with direct capture of cellular sgRNAs and changes in gene expression.

Image: 10X Genomics

Single cell full length RNA sequencing

In contrast to 3’ or 5’ scRNA sequencing, single cell full length RNA sequencing covers the entire sequence of RNA molecules, allowing for example allele resolution, as well as the identification of splice isoforms and genetic variants.

Single cell full length transcriptomics at the Custom Single Cell Omics platform

Two single-cell whole RNA sequencing technologies, Smart-seq3 and more recently FLASH-seq, are available on the Custom Single Cell Omics platform. These technologies quantify thousands of RNA molecules per cell, offering significantly greater sensitivity than the previous Smart-seq2. The choice between Smart-seq3 and FLASH-seq will be made according to the project and in discussion with the platform. The advantage of FLASH-seq is that it can be combined with ATAC-seq or scCNV in the same cells, thus obtaining single-cell multiomics data.

Starting material should count at least a hundred cells per sample.

Single cell full length transcriptomics at the NGS platform

The development of an approach combining 10X Genomics single-cell transcriptomics and Long Read sequencing techniques, by PacBio or Oxford Nanopore Technologies, is underway at the NGS platform. This unique approach available at the NGS platform generates reads covering the entire transcript (from the 5' to 3' end), enabling isoforms to be studied precisely, without the need for assembly.

5’ scRNA-seq+ VDJ

5’ scRNA-seq allows to quantify gene expression and to study the immune repertoire. In 5’ scRNA-Sequencing, poly(A)+ mRNAs are reverse transcribed using a polydT primer. However, in contrast to 3’ scRNA-Sequencing, the sequencing barcodes are not adjacent to the polydT primer, but they are located at the 5' end of the transcripts. The major advantage and use of the 5′ is to combine gene expression profiling with the identification of the full V(D)J fragment (a and b chain of TCR, heavy and light chain of Igs and BCR in B-cells) in the same cells.

5' scRNA-seq at the NGS platform

5’ scRNA-seq is available in routine at the NGS platform using the 10X Genomics pipeline. The required input material is 1,000 to 20,000 cells or nuclei and the recovery rate (yield) is around 50% of loaded cells/nuclei. The sequencing recommendations are around 50-100k reads per cell or nucleus.

Using feature barcodes, 5’ scRNA-seq can be combined with cell surface proteins expression using specific antibodies at single cell level (CITEseq) or to increase the multiplexing capacity (Cell Hashing). 

Image: 10X Genomics

Single cell DNA sequencing (scCNV)

Single cell DNA sequencing is used to identify DNA copy number variation. In this protocol, nuclei are subjected to protein degradation, followed by Tn5 labeling and PCR amplification, without any purification step. The resulting DNA is then sequenced. The protocol may or may not be combined with FLASH-seq sequencing of cytoplasmic RNAs from the same cell. Copy numbers can then be compared with gene expression levels.

Single cell DNA sequencing at the Custom Single Cell Omics platform

Single-cell single-cell DNA sequencing, with or without RNA sequencing, is currently being validated on the Custom Single Cell Omics platform, and will be available in 2025.

Single cell epigenomics is the study of the epigenome at the single cell level. It allows to study for example chromatin compaction and histone marks in each individual cell and to make a direct correlation with gene expression levels. The epigenetic heterogeneity within a population of cells can thus be assessed, and rare epigenetic events can be identified. The single cell initiative proposes several approaches:

Single cell ATAC-seq

Single Cell ATAC-seq, in which ATAC stands for “Assay for Transposase Accessible Chromatin”, allows to measure chromatin accessibility at the single cell level. The assay relies on the transposase Tn5, which cuts accessible chromatin regions and loads NGS adapters on the DNA fragments for subsequent sequencing. Single cell ATAC-seq provides insights into cell types and states, as well as gene regulatory mechanisms.

Single cell ATAC-seq at the NGS platform

Single cell ATAC-seq is available at the NGS platform using the 10X Genomics solution. The required input material is 1,000 to 20,000 nuclei and the recovery rate (yield) is around 50% of loaded nuclei. The sequencing recommendations are around 25k reads per nucleus.

Image : 10X Genomics

Single cell ATAC-seq at the Custom Single Cell Omics platform

A plate-based scATAC-seq approach is underway on the Custom Single Cell Omics platform. This approach is particularly suited for projects that are not compatible with 10X Genomics solutions, for example due to insufficient cell numbers.

Single cell ChIP-seq and Single cell CUT&Tag

Single-cell chromatin immunoprecipitation (scChIP) and, more recently, single-cell Cleavage Under Targets and Tagmentation (scCUT&Tag) allow determining at the single-cell level how specific chromatin marks are distributed across the genome. Marks associated with actively transcribed chromatin, such as H3 lysine 4 trimethylation (H3K4me3), or associated with gene repression, such as H3 lysine 27 trimethylation (H3K27me3) can be mapped in each cell and provide information about cell types and states, chromatin organization and gene expression regulation.

Single cell ChIP-seq at the Custom Single Cell Omics platform

The Custom Single Cell Omics platform implements scCUT&Tag and scChIP-seq for H3K4me3 and H3K27me3 on fresh samples. The scCUT&Tag can be combined with cytoplasmic RNA sequencing of the same cells, enabling the integration of single-cell epigenetic and transcriptomic data.

Combined scATACseq/RNA-seq

Combined scATACseq/RNA-seq allows to simultaneously profile gene expression and open chromatin regions, and thus to integrate two complementary layers of (epi)genomic information in each single cell. It thus provides a powerful tool to correlate chromatin accessibility with gene expression.

Combined scATAC-seq/RNA-seq at the NGS platform

Combined sc ATAC-seq/RNA-seq is available at the NGS platform using the 10X Genomics solution. The required input material is 1,000 to 20,000 nuclei and the recovery rate (yield) is around 50% of loaded nuclei. The sequencing requirements are aound 25k read pairs per nucleus.

Image : 10X Genomics

Combined scATAC-seq/RNA-seq at the Custom Single Cell Omics platform

The Custom Single Cell Omics platform is developing a plate-based approach combining ATAC-seq and FLASH-seq to measure both chromatin accessibility and gene expression in the same cells.  This approach is particularly suited forprojects that are not compatible with 10X Genomics solutions, for example due to insufficient cell numbers.

Conventional Cytometry

Conventional cytometry is based on the measurement of fluorescence in targeted channels following excitation by one or more lasers. This technique allows the expression of up to 50 markers to be measured and correlated with each other.

Conventional cytometry at the cytometry platform

Nine conventional cytometers are available on the cytometry platform. Having different (max 5 lasers and 28 parameters) but complementary characteristics and configurations, the choice of use is made according to the applications.

Image : Biolegend

Spectral cytometry

Spectral cytometry has unique optical collection and analytical capabilities. While in traditional cytometry mirrors and filters are used to separate narrow bands of light to individual detectors, in spectral cytometry the full emission spectrum is captured.  Spectral cytometry thus allows to simultaneously investigate a larger number of parameters than with conventional cytometry.

Spectral cytometry at the cytometry platform

Aurora spectral cytometera are available on the cytometry platforms of Paris and St Cloud. Their configuration allows the investigation of a panel of 50 different markers and automated substraction of autofluorescence.

Image : Cytek

Imaging cytometry

Imaging cytometry combines features of flow cytometry with microscopy. It allows the integration of morphological and fluorescent data at a single-cell level.

Imaging cytometry at the cytometry platform

The ImageStream X MKII (IMSX) technology is available at the Cytometry platform. This innovative technology allows acquisition of up to 12 channels (4 lasers: 405nm, 488nm, 561nm and 638nm), the ImageStream is equipped with 2 cameras, imaging every cell with high resolution (magnification up to 60X). The IMSX combines single cell morphological information provided by standard microscopy (shape, localization, cell cycle, etc) with large sample size and statistical robustness.

Spatial omics encompass a range of technologies that can be used to obtain -omics data on tissues while retaining spatial localization information. Beyond the identification of molecular subpopulations of cells, these approaches also provide information on the location of these identified subpopulations within the tissue of origin, their proximity to each other and to the extracellular matrix, blood vessels and other tissue components.

 The Single Cell Initiative has set up several spatial technologies, in collaboration with the experimental pathology platform (“PATHEX”, Renaud Leclère and André Nicolas):

Spatial transcriptomics

Spatial transcriptomics is a revolutionary technology that measures gene expression in tissue and maps its position within the tissue. It combines mRNA analysis with morphological context in intact tissue sections, offering an unpreceded view of tissue biology.

Sequencing based spatial transcriptomics

The Single Cell Initiative and PATHEX have implemented 10X Genomics' Visium and Visium HD technologies. They rely on sequencing for their read-out and they offer the advantage of whole transcriptome analysis. Resolution is around 55 microns (Visium) or 2 to 8 microns (Visium HD).

In these technologies, frozen or FFPE tissue sections are placed on top of a slide containing millions of capture oligonucleotides with spatial barcodes unique to each location. This step is performed using a dedicated equipment, the CytAssist from 10X Genomics. After tissue permeabilization, the mRNAs are released, bind to the capture oligos and are reverse transcribed. The cDNAs generated are then used to build Illumina libraries which are sequenced at the NGS platform. Gene expression data are superimposed on a high-resolution image of the tissue section, enabling visualization of the expression of mRNA within the tissue morphology.

Image: 10X Genomics

Imaging-based spatial transcriptomics on the Cytometry platform

The Single Cell Initiative has acquired a Xenium from 10X Genomics. This is a new imaging-based transcriptomics system that enables hundreds of RNA species to be visualized at the molecular level in their tissue context. The system is based on in situ hybridization and amplification of a targeted panel of genes of interest, which are then read by sequential imaging series. Gene panels range in size from 50 to 5000 genes and can be fully customized, standard for a tissue of interest, or a combination of both. Brightfield images (such as H&E) or immunofluorescence images can be taken on the same sections at a later stage to obtain a deeper tissue context.

Xenium's strength lies in its sub-cellular resolution and high sensitivity for rare transcripts and rare cells. What's more, a slide analyzed on the Xenium can then be analyzed in Visium, Visium HD or on the Orion (spatial proteomics), offering the possibility of spatial multi-omics.

You can download the flyer here!

In complement to this commercial approach, Antoine Coulon and Kyra Borgman are developing an in-house multiplexed FISH approach, which will be particularly suited for projects that are not compatible with commercial solutions or do not require high multiplexing.

Spatial proteomics

Spatial proteomics combines the detection of multiple proteins, usually using specific antibodies, with tissue microscopy. It enables the integration of morphological and protein expression data at the single-cell level.

Spatial proteomics on the cytometry platform

The cytometry platform proposes the Orion technology from Rarecyte, a tissue imaging system using multiplexed immunofluorescence (currently up to 18 markers). This system enables to establish complete phenotypic profiles and characterize whole tissue sections, in just a few hours and at sub-cellular resolution. It is based on a non-iterative staining method, meaning that all proteins are stained at once, saving time and avoiding tissue damage due to stripping. Brightfield images (such as H&E) can be taken on the same sections.

A Nanozoomer slide scanner is also available for optimizing tissue staining protocols, or for projects that do not require multiplexed staining.

Lung cancer tissue imaged with the Orion

Cytometry platform, Institut Curie

The bioinformatics platform is an integral part of the Single Cell Initiative, with several main missions.

Quality controls and first-level analysis

For each single-cell application, our team of bioinformaticians designs dedicated bioinformatics pipelines in order to check the quality of the data, and to provide a first level analysis. The quality controls are set up in close collaboration with the Custom Single Cell Omics and the sequencing platforms in order to highlight the key metrics of the experiments, and are summarized in a user friendly report.

These bioinformatics pipelines are run automatically when the data are collected from the sequencing platform, delivering the results in a couple of hours. In addition, all these pipelines are shared (https://gitlab.curie.fr/sc-platform) and are available for any collaborators within (and often outside) the institute. They are all developed following FAIR (Findable, Accessible, Interoperable, Reusable) principles, are documented, and packaged to ease their usage.

Experimental design and advanced analysis

In addition to standard quality controls and first-level analysis, the bioinformatics core facility can follow your project at all steps of the process. Before starting the experiment, we will be happy to discuss your experimental design with you in order to be sure that you will be able to (statistically) answer your biological questions. If needed, the bioinformatics core facility can also take the downstream analysis of your project in charge by dedicating a bioinformatician to your project for a given time. In the same way, co-supervisions are very welcome between the core facility and research teams in order to share expertise and knowledge on the field.

Training and networking

Finally, the bioinformatics core facility is involved in several networks dedicated to single-cell data analysis, covering many different aspects of the field from data processing to advanced statistical methods. As an example, we are part of the sinCellTE working group which organize every year a single-cell workshop dedicated to bioinformaticians and analysts in the field.