GSM8850466: WT-sham-3; Mus musculus; RNA-Seq
Hippocampal tissue was isolated from transgenic mice expressing tdTomato fluorescent protein in neurons. Following cardiac perfusion with saline, the brain was rapidly extracted, and the hippocampus was dissected. The hippocampal tissue was washed with ice-cold Hanks' Balanced Salt Solution (HBSS) to remove debris. The cleaned tissue was then transferred into a trypsin-containing solution, finely minced, and incubated at 37°C with gentle shaking for 15 minutes to dissociate single cells. To terminate digestion, DMEM supplemented with 10% FBS was immediately added. The cell suspension was then filtered through a 40-µm cell strainer and centrifuged at 300 g for 5 minutes to collect cells. The pelleted cells were resuspended in PBS containing 1% BSA and subjected to FACS using a BD Melody cell sorter. The cell suspension was loaded into the sample chamber, and tdTomato fluorescence was detected using the PE (Phycoerythrin) channel. Before sorting, forward scatter (FSC) and side scatter (SSC) thresholds were optimized in the flow cytometry software to distinguish live cells from dead cells and debris. A fluorescence threshold was set in the PE channel to differentiate tdTomato-expressing cells from non-expressing cells. Live tdTomato-positive cells were automatically sorted and collected into a collection tube containing culture medium for further analysis. The libraries were sequenced on an llumina Novaseq 6000 platform and 150 bp paired-end reads were generated.