GSM8621016: 49TH: 18 months, total homogenate; Mus musculus; RNA-Seq
Synaptosome samples were processed for RNA extraction according to (Smalheiser et al., 2014), and RNA was then extracted from the appositely prepared synaptosome samples and from TH samples through acidic phenol-chloroform extraction according to (Chomczynski, 1993). The synaptosomes were diluted in synaptosomal RNA buffer (50 mM Hepes, pH 7.5, 125 mM NaCl, 100 mM sucrose, 2 mM K acetate, 10 mM EDTA) containing protease inhibitors (1 tablet/10 mL, Roche, Cat. N. 11836170001), 160 U/mL Superase-In (Invitrogen, Cat.N. AM2694), 160 U/mL Rnase-OUT (Invitrogen, Cat. N. 10777019), quickly pelleted at 20.000 g for 20 min and rinsed twice in 4 × volume of synaptosomal RNA buffer and spundown again 20.000 g for 20 minutes. They were then resuspended in 100 μL of synaptosomal RNA buffer and RNA was extracted with standard Trizol protocol (Chomczynski & Sacchi, 1987) using Qiazol lysis reagent (Qiagen Cat. N. 79306). Libraries were prepared from 10 ng of input material (total RNA) using the SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (Takara, Cat. N. 635006) following the manufacturer's instructions.