Add a spacial dimension to your research
Single-cell RNA sequencing yields great insight in genome-wide transcriptomics. However, the tissue dissociation that is needed to obtain single cells, causes the loss of spacial information. TOMO-seq (Juncker et al. 2014) combines genome-wide transcriptomics with spacial information by sequencing microdissections of the desired tissue. We use a CEL-seq based approach to perform RNA-sequencing on the slices in a 96-well plate based system. Are you interested in doing Tomo-seq experiments and do you have access to a cryosectioner, do not hesitate to contact us.
Step 1: Sectioning of the tissue
Slicing of the tissue is done using a cryotome. Freezing of the tissue enables generation of slices of just a few micrometers thick. Depending on the tissue, TOMO-seq experiments can be designed in such a way that multiple body-axes can be followed with the slicing. The freezing of the tissue also preserves the quality of the RNA of the tissue. Every slice will get it’s own primer to ensure we can trace back which transcripts came from what slice. Above a cartoon example of a C. elegans that is sliced along the length of the body. Extra measures can be taken to fully stretch the animal.
Step 2: Processing of the RNA material
Every slice is put in a seperate well of a 96-well plate. The plate is prepared with mineral oil and has a microliter of a CEL-seq style primer. The primer contains a T7 promotor, a UMI, a slice-specific barcode and the poly-T tail that attaches to the mRNA molecules. The plate with slices are added to the plate it is stored at -80 Celcius. The protocol then starts with dispension of reagents using the GC Biotech’s Nanodrop II. For the generation of the ready-to-sequence transcriptome library, view the SORT-seq page, where the protocol is explained in more detail. The library is then sequenced on a Illumina Nextseq 500 using a 1×75 bp kit.
Step 3: Data analysis & -visualisation
The mapped TOMO-seq data can be used to find spatial transcription patterns along the body axis. For example, gene expression patterns known from literature can be used to find similar patterns of unknown genes, or new patterns can be found. Other applications of TOMO-seq experiments are following gene expression patterns over the development of embryos or following the patterns during regeneration of a tissue after damage. Above is a cartoon example of different gene expression patterns found over the body axis of a C. elegans