title: GSM7210821: b-TBI; Mus musculus; RNA-Seq
library_name: GSM7210821
library_construction_protocol: After 48 hours of injury, 4% isoflurane was used for anesthesia, followed by 1-2% isoflurane for maintenance anesthesia.  The mouse brain tissue from the hippocampal region was micro-dissected (n=3/group) after cardiac perfusion of 20-25 ml of PSB treated with hypothermia until the mouse liver turned pale.  The brain tissues were washed in pre-chilled PBS to remove any residual blood and placed in liquid nitrogen for rapid freezing before being transferred to -80°C for subsequent experiments. The frozen hippocampus was treated with NLB buffer (X-100 (Sigma-Aldrich, St Louis, MO, USA), 0.2 U/μL RNase Inhibitor (Takara, Kyoto, Japan), 250 mM sucrose, 10 mM Tris-HCl, 3 mM MgAc2, 0.1% Triton, 0.1 mM EDTA) for purification of nuclei using density gradient centrifugation with sucrose solutions of varying concentrations.   Initially, the lysate was diluted in 3 mL sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM MgAc, 0.1 mM EDTA, 1 mM DTT) and filtered through a 40 μm filter.   The filtered lysate was then centrifuged at 3200 × g for 10 minutes at 4°C.   The pellet was resuspended in sucrose buffer and incubated on ice for 2 minutes.   Subsequently, 12.5 mL of density sucrose buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM MgAc, 1 mM DTT) was layered under the sample.   The mixture was centrifuged at 3200 × g for 20 minutes, and the supernatant was discarded immediately.   The nuclei present on the side of the tubes were resuspended in 100 μL of PBS containing 0.04% BSA and 0.2 U/μL RNase Inhibitor (Takara, Kyoto, Japan).   Finally, the concentration of nuclei was adjusted to 1000 nuclei/μL suitable for snRNA-seq. Single-cell RNA sequencing (scRNA-seq) was performed using 10X Genomics Chromium platform. Related operations including Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT cleanup, complementary DNA amplification and library construction were all carried out following the manufacturer's protocol. Clustering analysis, proportion analysis, RNA velocity, myeloid trajectory analysis lymphoid trajectory analysis was performed.150-base-pair paired-end reads was applied.