Illumina HiSeq 2500 paired end sequencing; Transcriptome responses to dexamethasone of the hypothalamus of pigs with different genotypes at the GRAla610Val mutation
The experiment procedures took place at approximately the same time (10:00 to 12:00 AM). Three hours after the treatment  of dexamethasone,  the animals were anesthetized by an intravenous (i.v.) administration of a combination of ketamine (Ursotamin; Serum-Werk Bernburg, Bernburg, Germany) and azaperone (Stresnil; Janssen-Cilag, Neuss, Germany), and euthanized by an i.v. administration of T61 (Intervet, Unterschleißheim, Germany). Tissue samples were quickly dissected (<10min), frozen in liquid nitrogen, and stored at -80ºC. The experimental protocol (7221.3-1-024/16) has been approved by the Animal Care Committee of the Leibniz Institute for Farm Animal Biology and the State Mecklenburg-Western Pomerania (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei). 11 or 22 are genotypes of the pigs at the NR3C1 c.1829C>T (rs335303636, SNP) which 11 stands for homozygous CC and 22 stands for TT (Reyer et a. 2016; PMID 27736993) Total RNA was isolated using TRI reagent (Sigma-Aldrich, Taufkirchen, Germany). DNA traces were removed by DNaseI-treatment (Baseline-Zero DNase; Biozym, Hessisch Oldendorf, Germany), and the RNA was cleaned-up using the RNA Clean&Concentrator-25 Kit (Zymo Research, Freiburg, Germany). RNA quantity was determined using a Qubit fluorometer (ThermoFisher Scientific, Germany), and integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).  2 µg of total RNA with RIN > 8 was used for library preparation using a TruSeq Stranded mRNA Kit with 11 cycles of PCR-amplification according to the manufacture's recommendation (Illumina).