Ribosome-bund RNA-Seq of mus musculus: adult male nucleus accumbens
AAV-TRE-3g-Cre and AAV-FLEX-NBL10 were bilaterally injected into the NAcSh of Fos-tTA mice. Three weeks later, NAcSh cocaine- or saline-ensembles were labeled in cocaine-SA or saline-SA training. 1 day or 30 days after cocaine-SA or saline-SA training, the brain sections containing the NAcSh were quickly manually dissected and immediately homogenized in ice-cold supplemented hybridization buffer (in mM) (25 Tris [pH = 7.0], 25 Tris [pH = 8.0], 12 MgCl2, 100 KCl, 1% Triton X-100, 1 DTT (646563-10X, Sigma), 1Protease inhibitors (04693159001, Roche), 200 unit/ml RNase inhibitor (N2112S, Promega), 100 g/ml cycloheximide (14126, Cayman), 1 mg/ml heparin (H3393, Sigma-Aldrich). Homogenates were centrifuged for 10 min at 10,000 rpm, 4. RNA was extracted from 10% of cleared supernatant as input. The remaining supernatant lysate (output) was incubated with 3 g Rabbit anti-HA (H6908, Sigma-Aldrich) for 4 hours and 100 l Dynabeads Protein G (10003D, Novex) overnight at 4 with end-over-end rotation sequentially. Beads were collected on a magnetic rack, and washed three times with high-salt polysome wash buffer (in mM) (25 Tris [pH = 7.0], 25 Tris [pH = 8.0], 12 MgCl2, 300 KCl, 1% Triton X-100, 1 mM DTT, 100 g/ml cycloheximide). Purified mRNA was eluted from the Dynabeads using SuPerfectRI Total RNA Isolation Reagent according to the manufacturers instructions with the inclusion of a DNase digestion step. Agilent RNA 6000 Pico Kit (5067-1513, Agilent, USA) and Agilent 2109 bioanalyzer were used to evaluate the quality and concentration of purified mRNA.