title: Illumina NovaSeq 6000 paired end sequencing; Spatial transcriptomics of the axolotl forebrain
design: Spatial transcriptomics of the axolotl forebrain
library_construction_protocol: Whole axolotl brain was flash frozen before it was embedded in a prechilled optimal cutting temperature compound. The sample was then set into a dry ice bath with isopentane until frozen and stored at -80°C. Cryosections were cut at a thickness of 10µm, adhered to Visium (10x) spatial transcriptomic (ST) slides (10x) and stored at -80°C until the following day. Tissue slices were fixed in cold methanol before being stained with hematoxylin and eosin. ST slides were imaged as recommended on a Nikon T2i at 20x using a tile scan over all slice sections. Optimal permeabilization conditions were determined by using the Tissue Optimization kit (10x), and the optimal time was found to be 52 min. Spot-captured RNA was reverse transcribed before second-strand synthesis and cDNA denaturation. qPCR was used to determine the optimal number of cDNA amplification cycles as recommended by the manufacturer. cDNA was amplified using 18 cycles before continuing to Visium spatial gene expression library construction.