GSM7437659: TN3; Rattus norvegicus; RNA-Seq
OCT-embedded rat trigeminal nerve were cryosectioned coronally to a thickness of 10 μm. Tissue sections were layered onto the visium spatial tissue optimization slide containing oligonucleotides for mRNA capture (10x Genomics, Pleasanton, CA, USA).A Master Mix containing reverse transcription (RT) reagents and fluorescently labeled nucleotides is added on top of the tissue sections, resulting in fluorescently labeled cDNA synthesis. Tissue is enzymatically removed, leaving behind fluorescent cDNA covalently linked to oligonucleotides on the slide. Fluorescent cDNA is visualized under fluorescence imaging conditions verified using the Visium Imaging Test Slide. Fluorescent cDNA is visualized under fluorescence imaging conditions verified using the Visium Imaging Test Slide. H&E and fluorescence images are compared. The permeabilization time that results in maximum fluorescence signal with the lowest signal diffusion is optimal. The polyadenylation mRNA released by the covered cells is captured by the primers of the lattice. RT Master Mix containing the reverse transcription reagent was added to the penetrating tissue slice. After incubation with the reagent, full-length cDNA with spatial barcodes generated from polyadenylation mRNA on a slide can be obtained. The second strand synthesis Mix reagent is added to the tissue section area on the slide to initiate second strand cDNA synthesis. After the cDNA was transferred from the slide, the full-length cDNA was amplified by PCR and sufficient amount was obtained for the construction of the library. The cDNA was enzymatically cleaved and fragmented. DNA library was constructed by End Repair and A-tail. The constructed library was sequenced using lllumina novaseq 6000.