GSM9513049: N5721; Mus musculus; RNA-Seq
The mice were anesthetized and were performed a trans-cardiac perfusion with ice-cold N-methyl-D-glucamine-artificial cerebrospinal fluid (NMDG-ACSF) containing the following components (in mM): 93 N-methyl-D-glucamine, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 D-glucose, 5 sodium ascorbate, 2 Thiourea, 3 Sodium pyruvate, 10 MgSO4, 11.9 N-acetyl-L-cysteine, 1 CaCl2, and 6 ml HCl (pH 7.35–7.45), continually bubbled with O2 and CO2 (95% O2/5% CO2, v/v). Coronal brain slices (400 μm thick) containing regions of the mPFC were cut using a vibratome (Leica VT1000S, Germany), then promptly transferred to ice-cold NMDG-ACSF and incubated for 15 min. TTX (1 μM), CNQX (20 μM; Sigma-Aldrich, Catalog no. C239) and D-APV (100 μM; Sigma-Aldrich, Catalog no. A8054) were added in this step. The micro-dissected tissues were promptly frozen in liquid nitrogen and then stored at -80 ℃ until use. Frozen tissues were softened in the A buffer, and sieved through a 70 μm cell strainer. Triton X-100 was added to the sample homogenate to achieve a final concentration of 0.1%. The sample was on ice for 5 min incubated inutes, and then large impurities were removed using a 40 μm cell strainer. After centrifugation, 1 mL of C Buffer was added to the precipitate, and the cell nuclei precipitate was gently resuspended. Then, 500 μL of G2 buffer was slowly added to the bottom of the LoBind Tube, and 500 μL of G1 buffer was slowly and gently added to the upper layer of the G2 buffer without disturbing the boundary liquid surface. Resuspended cell nuclei were slowly added to the G1 buffer surface. After centrifugation, the cell nuclei aggregated at the bottom of the tube. The cell nuclei were resuspended in an appropriate amount of 1 × PBS with 0.5% BSA. Then, dissociated single nuclei were stained with AO/PI for quantity and agglomeration rate assessment using Countstar Fluorescence Cell Analyzer. The concentrations of the nuclei were adjusted to approximately 1,000 nuclei/μL for snRNA-seq. The snRNA-seq libraries were generated using the 10×Genomics Chromium Controller Instrument and Chromium Single Cell 3' V3 Reagent Kits (10×Genomics, Pleasanton, CA). Briefly, nuclei were concentrated to approximately 1000 nuclei/µL and loaded into each channel to generate single-cell Gel Bead-In-Emulsions (GEMs). After the RT step, 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. The final libraries were quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific) and the size distribution of the libraries were determined using a High Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). All libraries were sequenced by DNBSEQ-T7 on a 150 bp paired-end run.