GSM8263301: SM-J39ZI_S541_E1-50; Mus musculus; RNA-Seq
Two 8-week-old Snap25-IRES2-Cre;Ai14 mice (one from each sex) were perfused with artificial cerebrospinal fluid comprised of CaCl2 (0.5 mM), glucose (25 mM), HCl (96 mM), HEPES (20 mM), MgSO4 (10 mM), NaH2PO4 (1.25 mM), myo-inositol (3 mM), N-acetylcysteine (12 mM), NMDG (96 mM), KCl (2.5 mM), NaHCO3 (25 mM), sodium L-ascorbate (5 mM), sodium pyruvate (3 mM), taurine (0.01 mM), and thiourea (2 mM), bubbled with carbogen (95% O2 and 5% CO2). The pontine nucleus was dissected and embedded in 2% agarose, sliced into 250-µm sections with a vibratome, then subjected to enzymatic digestion with pronase (1 mg/mL) for 70 min at room temperature and trituration using fire-polished Pasteur pipettes to generate single-cell suspensions. Live single Pn neurons were isolated into 8-well strips containing SSv4 lysis buffer based on DAPI-/tdTomato+ expression using fluorescence-activated cell sorting, then stored at -80°C. To prepare single-cell transcriptome libraries, poly-adenylated RNAs were reverse-transcribed into full-length cDNA and subjected to 18 PCR amplification cycles according to the SSv4 protocol. Single-cell libraries were indexed and prepared for Illumina sequencing using the Nextera XT DNA Library Preparation Kit. Multiplexed libraries were sequenced on HiSeq2500 sequencers to generate 100-bp paired-end reads at a depth of 2.5 million reads per cell. SMART-seq v2 (SSv4; TakaraBio)