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?
How should I store TO/LP slides?
What tissue types can be used with the Spatial Transcriptomics technology?
Kidney tissue was initially resistant to optimisation and Spatial Transcriptomic interrogation
What quality control measures should I take before starting a Tissue Optimization experiment?
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?
Do I need to perform a Tissue Optimization experiment if I know the optimal permeabilisation and tissue removal conditions for my tissue of interest?
How big can my tissue section be?
Where can I find the Tissue Optimization Kit (discontinued) Safety Data Sheets?
USING IT
Can I use a fixative other than formaldehyde to fix my tissue?
Do I need to include the positive and the negative control in my Tissue Optimization experiments?
How should I image my H&E stained sections?
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?
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?
TROUBLESHOOT
Tissue was not entirely removed after tissue removal step.
Why can’t I detect a fluorescent signal at the end of my Tissue Optimization experiment?
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?
Are instructional videos available?
How should I store TO/LP slides?
How big can my tissue section be?
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?
How should I image my H&E stained sections?
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?
What is the recommended resolution for my images?
Does the reference total RNA used as the positive control need to be fragmented?
Should oligonucleotides used for library preparation be HPLC-purified or is purification by standard desalting enough?
RESULTS
What does a good library look like?
DNA HS
DNA 1000
What is the structure of the final library?
TruSeq Universal Adapter – barcode – UMI – cDNA -TruSeq Indexed Adapter.
Is the ST library strand-specific?
How long are the spatial barcode and UMI (Unique Molecular Identifier) sequences?
What sequencing platforms are the ST libraries are compatible with?
Illumina® NovaSeq
Illumina® HiSeq 3000/4000
Illumina® HiSeq 2500
Illumina® NextSeq 500/550†
Illumina® MiSeq
How deep should I sequence?
What settings should be used on a sequencer?
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 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.
GETTING STARTED
I have my two images and a pair of fastq files, how do I process & analyse the data?
You can find instructions for each tool at respective github repository under the links below