EVERYTHING YOU NEED
RESOURCES
Access detailed manuals and instructions on how to perform the protocol. Get guidelines on instrumentation and reagents for performing Spatial Transcriptomics in-house.
Tissue Optimization
Adapt the ST protocol to your specific fresh frozen tissue type
Library Preparation & Sequencing Prepare Spatial RNA-seq libraries from intact tissue sections.
Data Processing & Analysis
Data is intuitively processed and analysed with tailored bioinformatics tools.
Orders
Purchase our glass slides and perform Spatial Transcriptomics experiments in-house.
GETTING STARTED
Where can I find the Tissue Optimization Manual?
Are instructional videos available?
Cryosectioning of Samples for Spatial Transcriptomics: Link
How should I store TO/LP slides?
Store TO/LP blank slides at Room temperature, desiccated/low humidity.
What tissue types can be used with the Spatial Transcriptomics technology?
All tissues that express polyadenylated mRNA can potentially be analyzed using Spatial Transcriptomics. Human, rodent, and even plant tissues have been used successfully.
Kidney tissue was initially resistant to optimisation and Spatial Transcriptomic interrogation
Kidney tissue was initially resistant to optimisation and Spatial Transcriptomic interrogation
What quality control measures should I take before starting a Tissue Optimization experiment?
We recommend extracting RNA from your tissue of interest and analyzing its quality by Bioanalyzer. In order to ensure the best possible results, we recommend using tissue with a RIN (RNA Integrity Number) value of at least 7.
More details on how to collect samples for RNA extraction can be found in the “RNA Quality Requirements” section of the Tissue Optimization manual above.
How should tissue be frozen?
Tissue ischemia time should be kept to an absolute minimum prior to freezing in order to preserve RNA quality. A rapid freezing process should be used in order to avoid artifacts caused by ice crystal formation.
Do I need to perform a Tissue Optimization experiment if I know the optimal permeabilisation and tissue removal conditions for my tissue of interest?
The purpose of the Tissue Optimization experiment is indeed to find the optimal conditions for a tissue of interest. However, even if such conditions are known, we still recommend performing a Tissue Optimization experiment; especially if you are using Spatial Transcriptomics for the first time. Tissue Optimisation experiments are a cost effective way to confirm that the crucial elements of the Spatial Transcriptomics technique work in your lab, before moving on to more expensive Library Preparation experiments.
How big can my tissue section be?
A tissue section should be no larger than 6.3mm x 6.7mm. Tissue + OCT should fit within a 10mm x 10mm square.
Where can I find the Tissue Optimization Kit (discontinued) Safety Data Sheets?
You can download the Tissue Optimization Kit safety data sheets here
USING IT
Can I use a fixative other than formaldehyde to fix my tissue?
No, you should not use a fixative other than formaldehyde (3.6 – 3.8 % in PBS).
Do I need to include the positive and the negative control in my Tissue Optimization experiments?
We recommend including both controls in your Tissue Optimization experiment, especially if you are performing it for the first time. The negative control helps evaluate the efficiency of permeabilisation. If your experiment has failed, the positive control helps identify whether it was due to poor tissue quality or insufficient permeabilisation (no fluorescent cDNA signal in the tissue section wells but a bright signal in the positive control well) or due to reverse transcription failure (no signal in any of the wells).
How should I image my H&E stained sections?
In order to generate high resolution images of the stained tissue sections in a reasonable timeframe we recommend using a 10x or 20x objective with a high numerical aperture (NA) e.g. 10x NA 0.45, 20x NA 0.5, 20x NA 0.75.
At these magnifications many captured images (tiles) will be needed to reconstruct your entire tissue section.
Images can be collected manually, but we recommend using a motorized scanning microscope stage for a more efficient image capture.
How can I image the fluorescent cDNA footprint?
In microarray scanners (e.g. Innoscan 710) Cyanine-3 can be excited efficiently with the 532nm laser. When using a microarray scanner we recommend a resolution of at least 10 μm/pixel.
It is possible to capture the footprint with a standard fluorescent microscope. However, you will require a broad Cyanine-3/TRITC filter set (e.g. Chroma 49004; Zeiss 43HE), a high NA objective (e.g. 20x NA 0.75), and long camera exposure times.
RESULTS
What is the expected result of a Tissue Optimization experiment?
A successful Tissue Optimization experiment results in a fluorescent cDNA footprint that is readily distinguishable from the background and that mirrors the tissue morphology seen in the H&E image. Please see the sections “Introduction” and “Analysis” of the Tissue Optimization manual for examples of successful Tissue Optimization experiments. You can find the manual above.
TROUBLESHOOT
Tissue was not entirely removed after tissue removal step.
If tissue removal has failed and tissue remains on the slide, we recommend performing another Tissue Optimization experiment with added or prolonged tissue removal steps.
Why can’t I detect a fluorescent signal at the end of my Tissue Optimization experiment?
The fluorescent signal generated by the cDNA footprint of a successful Tissue Optimization is relatively weak compared to standard immunofluorescent samples. This means that successful experiments can appear not to have worked. However, the fluorescent cDNA footprint has an excellent signal to noise ratio, and can be easily imaged using standard equipment with the right instrument settings.
Optimal configuration for a widefield fluorescent microscope is a broad Cyanine-3/TRITC filter set (e.g. Chroma 49004; Zeiss 43HE), a high NA objective (e.g. 20x NA 0.75), and long camera exposure times.
GETTING STARTED
Where can I find the Library Preparation Manual?
You can download the manual guiding you through the Library Preparation here
Are instructional videos available?
How should I store TO/LP slides?
Store TO/LP blank slides at Room temperature, desiccated/low humidity.
How big can my tissue section be?
A single array of spots on an LP slide measures 6.5mm x 6.9mm (including frame spots). The 1007 active spots within the array cover an area of 6.1mm x 6.5mm (please see the Experimental Design section of the manual for a diagram).
In order to fit within the framing spots, tissue sections should not exceed 6.3 x 6.7mm. Tissue + OCT should fit within a 10mm x 10mm square.
USING IT
How many technical replicates should I include in my study using Spatial Transcriptomics?
Standard practice is to prepare libraries from at least 3 consecutive tissue sections of a given tissue block, and then choosing at least 2 of those libraries for sequencing.
How should I image my H&E stained sections?
In order to generate high resolution images of the stained tissue sections in a reasonable timeframe we recommend using a 10x or 20x objective with a high numerical aperture (NA) e.g. 10x NA 0.45, 20x NA 0.5, 20x NA 0.75.
At these magnifications many captured images (tiles) will be needed to reconstruct your entire tissue section. These are usually captured with the aid of a motorized scanning microscope stage.
How do I capture the array spots in my brightfield image?
The spots of the array are weakly stained by the H&E treatment. In order for this spot staining to be visible it is important to avoid overexposing your image. In fact a slight underexposure often yields the best results. i.e. your camera exposure time may need to be shorter than suggested by the “autoexpose” function in your imaging software.
What is the recommended resolution for my images?
Whilst this is to some degree dependent on the image quality and level of detail acceptable to the user, small, poorly resolved images can make data alignment difficult. Resolution will depend on the combination of microscope objective and camera used. We recommend using an objective with a numerical aperture (NA) of 0.4 or higher, and avoiding camera pixel binning if possible. We recommend saving your original microscope data files to avoid a situation where too much detail is accidentally lost during any image compression.
Does the reference total RNA used as the positive control need to be fragmented?
We recommend fragmenting the reference RNA to a fragment size of about 300 bp in order to approximate the experimental situation.
Should oligonucleotides used for library preparation be HPLC-purified or is purification by standard desalting enough?
The Cyanine-3-conjugated oligonucleotides (Cyanine-3 A Probe and Cyanine-3 Frame probe) should be HPLC-purified. We also recommend the aRNA Ligation Adapter to be HPLC-purified. All the remaining oligonucleotides can be purified by standard desalting.
RESULTS
What does a good library look like?
Bioanalyzer trace of a good library will appear as a single discrete peak approximating a bell curve and having an average size of around 300 to 600 bp.
DNA HS
DNA 1000
What is the structure of the final library?
The structure of the final library is as follows:
TruSeq Universal Adapter – barcode – UMI – cDNA -TruSeq Indexed Adapter.
Is the ST library strand-specific?
Yes, it is.
How long are the spatial barcode and UMI (Unique Molecular Identifier) sequences?
The spatial barcode is 18 nucleotides in length and the UMI is 7 nucleotides in length.
What sequencing platforms are the ST libraries are compatible with?
ST libraries are compatible with the following sequencing platforms:
Illumina® NovaSeq
Illumina® HiSeq 3000/4000
Illumina® HiSeq 2500
Illumina® NextSeq 500/550†
Illumina® MiSeq
Illumina® NovaSeq
Illumina® HiSeq 3000/4000
Illumina® HiSeq 2500
Illumina® NextSeq 500/550†
Illumina® MiSeq
How deep should I sequence?
We have found that sequencing to a depth of 100M read pairs per tissue section is a good start for many samples. Based upon sequencing saturation plots, one can decide to sequence further if needed.
What settings should be used on a sequencer?
Please use the following settings:
Library Preparation Kit: TruSEQ LT
Read Type: Paired End
Number of Indexes: 1, single-index configuration
i7 Index: 6 cycles
Read 1: 26 cycles minimum, 30 cycles are recommended
Read 2: 50 cycles (if you are using 75 cycle kit) or 120 cycles (if you are using 150 cycle kit)
Library Preparation Kit: TruSEQ LT
Read Type: Paired End
Number of Indexes: 1, single-index configuration
i7 Index: 6 cycles
Read 1: 26 cycles minimum, 30 cycles are recommended
Read 2: 50 cycles (if you are using 75 cycle kit) or 120 cycles (if you are using 150 cycle kit)
TROUBLESHOOT
According to my Bioanalyzer traces, there is no aRNA in my sample after in-vitro transcription.
If your Bioanalyzer trace shows no aRNA or peaks of aRNA of incorrect size, we recommend redoing your experiment. Please be careful performing the cDNA and RNA purification steps: do not over-dry the beads as it might impact nucleic acid recovery.
If a peak corresponding to very short aRNA fragments appear on the trace, please check the RNA quality in the tissue by extracting RNA from a few tissue sections and measuring its RNA Integrity Value (RIN) is described in the manual above.
To obtain aRNA with an average length between 200 and 500 nt, it is important to fix the tissue sections for 10 min with freshly prepared 3.6-3.8% formaldehyde solution.
My Bioanalyzer trace on the final library shows no peaks. I used the Bioanalyzer DNA 1000 kit.
Try to re-run the samples using Bioanalyzer DNA High Sensitivity kit.
GETTING STARTED
I have my two images and a pair of fastq files, how do I process & analyse the data?
We recommend to use the open source packages available on github.
You can find instructions for each tool at respective github repository under the links below
Where do I find the ID files for the st_pipeline?
You can find the ID files for the different LP slide batches here
USING IT
What settings should I use for the st_pipeline?
The defaults are usually sufficient for a first analysis, if you want to tweak the settings afterwards, please read the documentation on the open source st_pipline github repository (https://github.com/SpatialTranscriptomicsResearch/st_pipeline).
RESULTS
How many genes/molecules should I detect from a spatial transcriptomics experiment?
The results from your analysis varies depending on the tissue type, the size of the tissue section (how many of the spots were covered) as well as the experimental conditions used during library preparation.
TROUBLESHOOT
There is no spatial expression in my data, what is wrong?
Please check that you are using the correct ID file. If the wrong ID file was used for the initial data processing using the st_pipeline, the spatial resolution is lost. If the wrong ID file was used simply rerun the pipeline using the correct ID file.
PLACING AN ORDER
How do I place an order?
To place an order through 10x Genomics, request a quote from the product page.