title: GSM8864083: t-HIP; Mus musculus; RNA-Seq
library_name: GSM8864083
library_construction_protocol: Mice were sacrificed 48 hours post injury, and the hippocampus was micro-dissected (n=3 per group) and washed in precooled PBS to remove residual blood. Then, the tissue was flash-frozen in liquid nitrogen and transferred to -80 ℃ until further use.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) was used to homogenize the frozen tissue. Different concentrations of sucrose were applied to purify the nuclei using sucrose density gradient centrifugation. A final concentration of around 1000 nuclei/μL was used for scRNA-Seq. The scRNA-Seq libraries were built using the 10X Genomics Chromium Controller Instrument and Chromium Single Cell 3' V3.1 Reagent Kits (10X Genomics, Pleasanton, CA, USA). Briefly, the acquired cell nuclei were loaded into several channels to obtain single-cell Gel Bead-In-Emulsions (GEMs). After reverse transcription, barcoded cDNA was released from the GEM and then purified. After amplification, the barcoded cDNA went through fragmentation, ligation with adaptors, and amplified with index PCR. The Qubit High Sensitivity DNA assay (Thermo Fisher Scientific, Waltham, MA, USA) was applied to quantify the final libraries, and the size distribution was determined by the High Sensitivity DNA chip using Bioanalyzer 2200 (Agilent Technologies, Santa Clara, CA, USA). Novaseq6000 (Illumina, San Diego, CA, USA) was used for sequencing on a 150 bp paired-end run.
identifiers.namespace: GEO
identifiers.id: GSM8864083_r1