GSM7070557: Brain,3xDR, rep2, scRNAseq; Mus musculus; RNA-Seq
In brief, brains without cerebellum and olfactory bulb from 3xDR and control mice were harvested and cut into 1 mm-thickness sections using stainless steel brain matrices. Next, brain slides were incubated in 95% O2 and 5% CO2 bathed Choline Chloride Solution (92 mM choline chloride, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgSO4·7H2O, 0.5 mM CaCl2·2H2O, 12 mM N-acetyl-L-cysteine) with 10 μM NBQX and 50 μM APV and 2% FBS (Gibco, cat: l6140071) for about 30 minutes. After that, brain slides were digested in 20 U/mL papain and 100 U/mL DNase I (Worthington, cat: lK003176) for 30 min at 37 °C, followed by incubation with 1 mg/mL protease (Sigma, cat: P5147) and 1 mg/mL dispase (Worthington, cat: LS02104) at 25°C for 30 min with shaking. Next, brain slides were further tittered using a 1 mL pipette. Single-cell suspensions were then collected and filtered through 40 μm cell strainers (Falcon). The cell debris was removed by 30% Percoll (Millipore, cat: P1644) in Choline Chloride Solution. Finally, cells were resuspended in Choline Chloride Solution for further experiments. Approximately 10^7 cells were obtained from each brain. Approximately 24,000 cells for each mouse were loaded for 10x Genomics library preparation. Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation,so that each cell was paired with a bead in a Gel Beads-in-emulsion(GEM). After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA synthesis, each cDNA molecule was tagged on the 5'end (that is, the 3'end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Briefly, 10× beads that were then subject to second-strand cDNA synthesis, adaptor ligation, and universal amplification.Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3' end of the transcripts linked with the cell barcode and UMI. All the remaining procedures including the library construction were performed according to the standard manufacturer's protocol (CG000206 RevD). Sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). The libraries were sequenced on NovaSeq6000 (Illumina) using 2x150 chemistry.