GSM7519182: TBI 3 dpi mouse brain; Mus musculus; OTHER
Mice were subjected to controlled cortical impact injury or sham surgery (i.e., craniotomy only) as described previously. Injury parameters were: impact speed, 3.5 m/s; deformation depth, 1.0 mm; duration, 400 ms. Mice were sacrificed three days after TBI or sham surgery, their brains dissected in an RNase-free environment and immediately transferred into refrigerated O.C.T. compound (Sakura Tissue-Tek O.C.T. compound, Nagano, Japan) for flash-freezing in chilled isopentane. Samples were stored at -80°C until further processing. Library preparation of mouse brain samples was performed according to the Visium Spatial Gene Expression Reagent Kits User Guide (CG000239 Rev C, 10x Genomics, USA). Briefly, brain samples were cryosectioned until the dorsal hippocampus and dentate gyrus region were visible. Next, 10 μm sections were collected onto a pre-chilled Visium slide. All sections were dried onto the slides at 37°C for 1 minute, fixed in pre-chilled 100% methanol at -20°C for 30 minutes, and stained in Mayer's Haematoxylin for 5 minutes and Eosin for 2 minutes. Slides were then mounted in 85% glycerol for coverslipping and brightfield imaging (Axio Z1 slide scanner, Zeiss) For Visium samples, permeabilisation of mouse brain samples was carried out for 18 minutes, resulting in a cDNA library size of 470 bp post-fragmentation. Library quantification was carried out using the KAPA Library Quantification kit (Roche), followed by in-house sequencing using a high output reagent kit and NextSeq500 instrument (Illumina) at the Institute for Molecular Bioscience Sequencing Facility. Sequencing was performed using the following protocol: Read1 - 28 bp, Index1 - 10 bp, Index2 - 10 bp, Read2 - 120 bp. . For Legacy samples,  ST sequencing libraries were prepared as per the manufacturer's instructions, with pre-permeabilisation and permeabilisation performed at 20 minutes and 7 minutes, respectively. On-slide cDNA synthesis, tissue removal, probe cleavage, and final library preparation were all performed as per the manual. High-quality cDNA libraries with sizes ranging between 660-780 bp were obtained and sequenced on the Illumina Nextseq500, using a 150 cycles kit with a read configuration of read 1 (26 bp) and read 2 (124 bp).