GSM9370847: hippocampal tissue from Meth3; Mus musculus; RNA-Seq
After the mice had been exsanguinated via transcranial perfusion with physiological saline (PS, Thermo Fisher Scientific), hippocampal tissues for analysis were dissected. Briefly, the tissue was added to 1 ml of pre-chilled lysate, sheared into small pieces of approximately 1 mm3, and 2 ml of pre-chilled lysate was added. The tissue was transferred to a tissue homogenizer (885300-0007, Kimble Dounce Homogenizer) and ground slowly five times. Then the tissue was incubated on ice for 5 min. Subsequently, 4 ml of pre-cooled 2% BSA solution was added, and the process was terminated by pipetting with a barrel pipette. The samples were passed through a 30 μm cell sieve, and the filtrate was collected and centrifuged at 300×g for 5 min at 4 °C. The supernatant was discarded. The cells were resuspended in 5 ml of 2% BSA, centrifuged at 4 °C for 5 min at 300×g, and the supernatant was discarded again. The cells were then resuspended in 500 μL of resuspension solution (1×PBS, 2% BSA, 0.1% RNase inhibitor). The cells were stained with Propidium Iodide (PI) and sorted to remove debris and impurities using a BD FACSAria II flow cytometer. The sorted cells were collected and counted. Single-nucleus suspension was added to the 10×Chromium chip according to the instructions for the 10×Genomics Chromium Single-Cell 3' kit (V3), with the expectation of capturing 8000 nuclei. After the reverse transcription step, single-nucleus Gel Bead-In-Emulsions (GEMs) were broken, and barcoded cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed, ligated with adaptors, and index PCR amplified. Libraries were sequenced by LC-Biotechnology (Hangzhou, China) on an Illumina NovaSeq 6000 sequencing system (paired-end sequencing, 150 bp) at a minimum depth of 20,000 reads per nucleus.