GSM6998291: Purkinje cells, wild type, rep3; Mus musculus; RIP-Seq
Each cerebellum was homogenized using a dounce homogenizer in 1.5mL lysis buffer (1% NP-40, 100mM KCl, 50mM Tris pH 7.4, 12mM MgCl2, 0.1mg/mL cycloheximide, protease inhibitor cocktail (Sigma, 11836170001), 1mg/mL heparin, 5µL/mL RNAseOUT (ThermoFisher 10777019)). Ribosomes were then isolated as previously described (Sanz 2009) Briefly, lysates containing tagged ribosomes were incubated for 4 hours at 4˚C with 5µg rat anti-HA antibody (Roche, 11867423001) after which 400µL protein G-coupled dynabeads (Thermo Scientific, 10004D) were added to the lysate and incubated at 4˚C overnight. The following day ribosome-bound beads were washed three times with high salt buffer (0.3M KCl, 1% NP-40, 50mM Tris pH 7.4, 12mM MgCl2, 0.1mg/mL cycloheximide, 0.5mM DTT), and mRNA was isolated using buffer RLT and spin columns (Qiagen RNEasy Mini Kit). RNA was eluted in 30µL H2O Libraries were constructed using the Kapa Stranded mRNA-Seq Kit (#KR0960) in accordance with manufacteur's instructions.