title: GSM5639997: N135; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized with CO2 and transcardially perfused with cold Hank's balanced salt solution (HBSS) (without Ca2+ and Mg2+). Vagal ganglia were quickly dissected after perfusion in cold buffered HBSS (10 mM HEPES, pH7.2-7.5) and each was cut into 2~3 pieces. Ganglion tissue was then digested with papain solution at 20 U/ml (Worthington LS003124, in buffered HBSS containing 0.5 mM EDTA, 1.5 mM CaCl2, 0.4 mg/ml L-cysteine, pre-warmed for 30 min for activation) for 10 min at 37°C, and then collagenase/dispase solution (collagenase IV, 300 U/ml, Worthington LS004186; dispase, 1mg/ml, Thermo Fisher Scientific 17105-041, in HBSS, pre-warmed) for 30 min at 37°C with tube inversion to agitate every 10 min. After centrifuging at 400 g for 4 min, tissue pellet was resuspended in 1 ml L15 medium (with 10mM HEPES pH 7.2-7.5 and 10% fetal bovine serum) and triturated using fire-polished Pasteur pipettes. A Percoll gradient spin was used to clear axonal debris and glial cells by layering the cell suspension onto a buffered L15 medium (10 mM HEPES)/Percoll (4:1) solution and then centrifuging for 9 min at 400 g. The cell pellet was resuspended in 2 ml buffered L15 medium, centrifuged again for 3 min at 750 g, and resuspended in 200 µl suspension medium (L15 medium, 10mM HEPES pH 7.2-7.5, 1mg/ml BSA, and 0.1% Pluronic F-68). cDNA samples were prepared using SMART-Seq v3 Ultra Low Input RNA Kit (Clontech), and sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina FC-131-1096) and Index Adapters (Illumina, FC-131-1002), following the C1 system for mRNA Seq Protocol (Fluidigm).