Spatial Transcriptomics allows gene expression analysis of all mRNAs in a tissue section in a positional context. The SpaRTAN team - a 10X Genomics Certified Service Provider comprising a multi-disciplinary team across Newcastle University University’s core facilities - offer a comprehensive end-to-end service from sample preparation to bioinformatics analysis. The expertise of a very experienced team trained in tissue preparation and sectioning, image analysis, next-generation sequencing and bioinformatics combine to deliver a complete spatial transcriptomics analysis servuice using the Visium platform from 10X Genomics.
We can accept both flash frozen or OCT embedded tissue for frozen sample analysis or FFPE tissues for fixed tissue analysis (please contact us to find out more).
Technology and Service Overview
- Each tissue type is first assayed for RNA quality and usually only tissues with a RIN score of ≥ 7 are recommended to be taken forward for analysis.
- Each tissue type is tested in a separate experiment to determine the optimal permeabilization time to use on the Gene Expression slide. This will vary between tissues and species and needs to be done for every tissue type under investigation. For example, normal liver and liver tumour would require two optimisation experiments.
- Some tissues may require more optimisation than others - for example, skin, pancreas and bone.
Fresh-frozen 10µM sections from tissue embedded in OCT are placed onto a microscope slide containing four 6.5 mm X 6.5 mm capture areas. Each capture area contains 5000 spots; each spot consisting of a high density group of spatially barcoded probes that contain a sequencing adapter, a spatial barcode unique to that spot, a 12-base UMI, and a poly-T region designed to capture poly-adenylated mRNA.
- After placement, the section is fixed, H&E stained, and imaged for histological purposes - then the tissue is gently permeabilized, allowing the mRNA to be released and held in position by the capture probes.
- The probes are then removed from the slide and sequencing libraries are prepared from the capture RNA and sequenced (≥50,000 reads per capture spot).
- The processed Visium data is overlaid with the H&E image to determine which genes are expressed, and where those genes are expressed within the tissue section
- A report showing the spatial cell cluster analysis and a list of differentially expressed genes in each cluster is transferred from our server along with the raw data files to allow users specific analysis if need.
Frequently asked Questions
Spartan will take your tissue (or tissue provided by the HDBR or NovoPath tissue bank – see FAQ 6 below), perform quality control analysis and then run through the Visium workflow from 10X Genomics. We will deliver annotated outputs from the Space Ranger software showing cell cluster analysis on the imaged sections, a list of the most differentially expressed genes within clusters to allow for immediate interpretation of the data, as well as the raw data files for you to perform your own independent analysis.
Rapid freezing is essential to maintain Tissue morphology and RNA integrity. There are two methods to do this:
- Freeze tissue first, then embed in OCT
- Freeze and embed tissue at the same time
Method one: Snap freezing the tissue
- Fill a metal container 2/3 with isopentane
- Place isopentane into a second container of liquid nitrogen, to the same level as the isopentane in the metal container, cool for at least 15 minutes
- Use lab tissue to soak up any liquid around the sample (to stop ice crystal formation)
- Lower sample into isopentane on a spatula for one minute
- Immediately place frozen tissue sample in a sealed precooled container, sample must be sealed to prevent evaporation and dehydration, and store at -80°C.
- The tissue can be sent to us in this form and we will embed it in OCT.
Method 2: Simultaneously freeze and embed the tissue
- Prepare an isopentane, liquid nitrogen bath as Method 1 above.
- Dry the tissue
- Label a cryomold
- In a petri dish, cover the tissue with OCT then place it into the cryomold and fill with more OCT to cover the tissue making sure there are no air bubbles.
- Lower the mold into the precooled isopentane, but DO NOT SUBMERGE it, when the OCT is opaque, transfer to a sealed container and store at -80°C
The capture area of the Visium slides are 6.5 x 6.5mm. The tissue should not be bigger than approximately 5 x 5mm, for ease of handling during placement. The capture area is surrounded by a fiducial frame, and it is best to avoid placing the tissue on the fiducial frame as the tissue area outside the capture area will not generate data. In addition, the fiducials are used for alignment by the Space Ranger software. If the fiducials are covered, manual alignment may be required.
Depth of tissue is important; 0.5mm deep or more would be ideal, for ease of cutting, and to allow the removal of one or two 50 µm sections for quality control RNA extraction and QC. Alternatively, a separate representative frozen piece of the same tissue approximately 10-30ug may be provided for this purpose and will speed up the QC turn around.
The SpaRTAN team has an Illumina NovaSeq 6000 opperated by a dedicated team offering the opportunity to scale sequencing according to project size and is included in the cost of the service. The team is also experienced in designing and managing projects that require single cell library preparation and sequencing.
Provided the tissue passes QC checks you should have results in the form of an electronic report within 6-8 weeks of the project starting. The exact time a project commences will depend upon the workload of the team at that time. The sooner the tissue is received, the sooner it can join the queue of projects.
It is important that the experiment is designed correctly so that the specific biological questions under investigation can be tested. The SpaRTAN team will meet with any potential users prior to a beginning any project so that the requirements, scope and what outcomes would like to be gained from the experiment.
The data generated will be aligned and quantified using SpaceRanger. Further quality control checks followed will by an integrated clustering analysis of technical replicates in R. Finally, differential expression analysis will be performed to generate gene lists for each cluster. All of the outputs from SpaceRanger will be provided to the user including: Run summary HTML, run summary, processed images, run analysis, gene- barcode matrices MEX and HDF5, barcoded BAMs, and molecule info HDF5. A report of combined analysis report of all sections analysed in R will be provided, which will include QC metrics, combined clustering results and lists of differentially expressed genes between clusters. Finally, the raw data as FASTQ files for each section and Image Files will be provided along with the summary reports so that more in depth analysis can be made. Support for addition bioinformatics analysis can be provided at a hourly cost rate.
The team are also experienced in designing and managing projects that require single cell bioinformatic analysis and this can be discussed at eth pre-project meeting.
For Visium Gene Expression slides, the number of replicates you will need will depend on your research question. The number of replicates will vary depending on the tissue being processed, as well as how variable each consecutive section is from the last section. For a highly heterogeneous tissue block, there may be differences between each 10-micron section, even if they are consecutive sections. We recommend using at least 3 replicates per tissue type.
In addition, multiple biological replicates will also be necessary, the exact number of which depend on the individual experiment. We will discuss this with you prior to commencing your project.
Yes the HDBR can provide embryonic and fetal tissue. Go back to the home page for details, project registrations or email to: firstname.lastname@example.org
Tissue can also be provided through the Novopath tissue bank www.novopath.co.uk/bio-banking
The capture area is 6.5 x 6.5mm. There are a total of 4992 total spots per capture area and each spot is 55µm in diameter with a 100µm centre-to-centre distance between spots.
Each spot contains in the order of millions of probes.
The number of cells captured in a single spot is based upon the tissue type, cell size and section thickness, this is generally between 1-10 cells
This is an area the SPARTAN team are currently working on and can be discussed with the team during the pre-project meeting.
Yes, Newcastle University’s Bioinformatics Support Unit can provide additional support beyond the standard analysis provided through the SPARTAN service. Please can you contact us to discuss your specific project requirements.