RNA-Seq of Rattus norvegicus: Differentiated TH+ dopaminergic neurons
The 5 month old male Sprague Dawley rats, inhoused at the Small animal Facility of Department of Biotechnology, CUSAT were used in this study. Peripheral blood was collected by cardiac puncture and Peripheral Blood Molonuclear cells (PBMNCs) were isolated by density gradient centrifugation using Histopaque 10831. The PBMNCs were then seeded onto fibrin (2 mg/ml fibrinogen and 2 IU/ml thrombin) coated tissue culture plates and cultured for 12 days in Neurobasal - A medium (1X) Liquid GibcoTM (Catalogue No.10888022, Gibco)containing 1X Glutamax (Stock  100X) (Cat. No. 35050061), 2mM L-Glutamine (Stock  200 mM) (Cat. No. 25030081), 1X B27 (Stock  50X) (Cat. No. 17504044), basic Fibroblast Growth Factor (bFGF) (Cat. No. 100-18B, Peprotech), Cerebral Dopamine Neurotrophic Factor (CDNF) (Cat. No. 450-05, Peprotech) and 1% Penicillin-Streptomycin solution (Stock - 100X) (Cat. No. 15240096) at standarad conditions. The medium was changed every 2 days interval. On 12th day, culture medium was discarded and the cells were collected in RNA later solution (Cat No. AM7020, Sigma). On 12th day, RNA was isolated using RNA isolation kit (Cat.No. 83913, Sigma). The RNA concentration was assessed using Qubit assay. This was followed by RNA enrichment, RNA fragmentation, Random priming, cDNA synthesis and Library Preparation. Library Preparation was performed using NEB Ultra RNA Lib Prep kit.  Library size was assessed using the Agilent 2100 Bioanalyzer, concentration byQubit assay, and final quantitation by qPCR. The sample was then sequenced using Illumina HiSeq platform. The raw data obtained was then taken for RNA Seq analysis.