GSM9135910: F_D1_Pro_sal_IP_9; Rattus norvegicus; RNA-Seq
Tissue punches (2mm) collected from 200 µm sections of the nucleus accumbens of individual rats were used for subsequent processing. Immunoprecipitation of polyribosomes from D1- and D2-Cre rats was performed as follows: frozen bilateral tissue punches were homogenized by douncing in a homogenization buffer (2.5-4% w/v) containing 50 mM Tris-HCl (pH 7.4), 100 mM KCl, 12mM MgCl2, and 1% NP-40 Substitute supplemented with 1 mM DTT, 100 µg/mL cycloheximide, 200 U/mL RNasin Plus (Promega), 1 mg/mL heparin, and EDTA-free protease inhibitor (ApexBio). Homogenate was spun at 10,000 x g at 4°C for 10 min and cleared lysate transferred to new tubes. Approximately 6-8% of lysate was retained as input and frozen at -80°C until further processing. The remaining lysate was incubated overnight at 4°C with an anti-HA antibody (BioLegend, Anti-HA.11; 1:200 v/v) with gentle end-over-end rotation, followed by an overnight incubation at 4°C with magnetic beads (Pierce; Protein A/G). The following day, beads were washed at 4°C 3 times with a high-salt buffer containing 50 mM Tris-HCl (pH 7.4), 300 mM KCl, 12 mM MgCl2, 1% NP-40 Substitute, 0.5 mM DTT, and 100 µg/mL cycloheximide. Finally, genomic DNA removal and RNA purification were carried out using the RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer's protocol. Stranded RNA-seq libraries were prepared by the Florida State University NGS Library Facility following poly(A) mRNA purification from total RNA based on magnetic beads, cDNA synthesis using random hexamers, and final amplification using barcoded primers (New England Biolabs, Ipswich, MA, USA). The resulting barcoded and stranded libraries were quantified using a KAPA qPCR library quantification kit (KAPA Biosystems, Boston, MA, USA) and the absence of adapters or primers contamination was verified on an Agilent TapeStation (Agilent Technologies, Santa Clara, CA, USA).