GSM9377359: P21_THALAMIC_PLANE_REP2; Mus musculus; OTHER
The brains were simultaneously embedded in OCT compound and frozen for cryo-sectioning at a thickness of 10μm. The tissue preparation followed the protocol provided by the company 10X Genomics more specifically using the Visium Spatial Gene Expression (GEX) and Tissue Optimization (TO) Slide & Reagent Kits (catalog numbers 1000193 and 1000187). Brain sections were done on 3 different planes (anterior, thalamic and brainstem planes). The brain sections were thus placed on TO and GEX slides, and then stored at -80°C until further processing. The TO and GEX slides were then fixed in methanol and stained with hematoxylin and eosin (H&E). This helped with tissue morphology visualization using an automated slide scanner (Zeiss Axioscan.Z1). Tissue permeabilization time was optimized using the TO slides, and 30 minutes was selected as the ideal duration. The GEX slides were incubated with reverse transcription reagents (RT) and then followed a cDNA amplification of 15 cycles. The library construction was performed according to the manufacturer's protocol. This included steps of cDNA fragmentation, end repair, A-tailing, adaptor ligation, and sample indexing and amplification. A Bioanalyzer (Agilent) assessed the quality control of the constructed libraries. Finally, the Illumina NovaSeq S4 platform by Genome Quebec performed the sequencing. Genome Quebec also completed the base calling, demultiplexing, and FastQ file generation.