GSM7109822: mPFC_diestrus, scRNAseq; Mus musculus; RNA-Seq
The mPFC tissues were extracted using a brain matrix from two estrous female mice, two diestrous female mice, and two male mice. The dissected tissue was then cut into small pieces and transferred to a 15 ml tube containing 20 U/mL pre-activated papain (Worthington, LK003178, 37°C for 30 min) and 0.1 U/uL recombinant RNase inhibitor (Takara, 2313A). Papain digestion was performed at 34℃ for 20 min. Following digestion, the papain solution was replaced with ice-cold, carbogen-bubbled ACSF (pH 7.2-7.4, 125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 25 mM glucose, 1 mM MgCl2, and 1 mM CaCl2, oxygenated with 95% O2 and 5% CO2) containing 1% fetal bovine serum (FBS) and 0.1 U/μL recombinant RNase inhibitor. The tissue pieces were dissociated using gentle pipetting through 1000μL and 200μL tips, to obtain single-cell suspensions. Subsequently, the suspensions were filtered through a 35μm cell strainer (Corning, 352235) to remove debris, and then layered onto 30% Percoll (Sigma, P4937, diluted by ACSF containing 1% FBS and 0.1 U/μL recombinant RNase inhibitor), followed by centrifugation at 700g for 10 minutes at 4℃. After two washes, the cells were resuspended in 1X PBS with 0.02% BSA and 0.1 U/μL recombinant RNase inhibitor. The cell viability and concentration were determined, and 15000 single cells per sample were uploaded onto 10 × Genomics 3′ library chips, using the Single Cell 3′ Library & Gel Bead kit (v.3.1), as per the manufacturer's instructions. The cell viability and concentration were determined, and 15000 single cells per sample were uploaded onto 10 × Genomics 3′ library chips, using the Single Cell 3′ Library & Gel Bead kit (v.3.1), as per the manufacturer's instructions.The resulting libraries were sequenced on an Illumina NovaSeq 6000.