title: GSM9244188: Cerebral tissue，PBS treatment for three days after TBI, rep1; Mus musculus; RNA-Seq
library_construction_protocol: Transfer fresh Cerebral tissue into a culture dish (the dish is placed on moist ice), wash the tissue surface with 1X PBS to remove bloodstains, lipids, and other adhering substances. Cut the tissue into 0.5mm(2) pieces, transfer the tissue into a 15mL centrifuge tube, add 5mL enzyme solution (1mg/mL sigma collagenase I, 1mg/mL sigma collagenase II, 0.5mg/mL sigma neutral protease), react at 37°C in a water bath shaker (100rpm) for 30 minutes, then add 1× PBS (containing 10% fetal bovine serum FBS) to terminate digestion, and repeatedly pipette the mixture 5-10 times with a transfer pipette. Subsequently, filter through a 40μm cell strainer, collect the cell suspension in a 15mL centrifuge tube, centrifuge at 4°C, 500g for 50 minutes. After centrifugation, discard the supernatant, add 3 mL red blood cell lysis solution (MACS 130-094-183, 10×), incubate at room temperature for 5 minutes. After lysis, centrifuge at 4°C, 300g for 5 minutes, collect the cell pellet after centrifugation, remove dead cells using a Dead Cell Removal Kit (MACS 130-090-101), resuspend the cells in 1× PBS (0.04% BSA), and adjust the cell concentration to 700-1200 cells per μL. According to the 10x Genomics Chromium Single Cell 3' Reagent Kits (V3.1) manual, load the single-cell suspension onto a 10x Chromium chip, expecting to capture 8000 cells. Perform cDNA amplification and library construction following the standard protocol. The libraries are sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end sequencing, 150bp) by LC-Bio Technology Company (Hangzhou, China).
identifiers.namespace: GEO
identifiers.id: GSM9244188_r1