Pituitary Gland RNA Extraction and Sequencing

The entire pituitary gland of each animal was homogenized in 1 mL of TRIzol reagent (Invitrogen, Karlsruhe, Germany). Total RNA was extracted in accordance with the manufacturing instructions. The extracted RNA was additionally treated with DNase I using the RNase-free DNase kit (Base-Zero DNase, Biozym, Hessisch Oldendorf, Germany) and with the Zymo^® RNA Clean&Concentrator kit (Zymo Research, Irvine, CA, USA). The RNA quality was assessed using an Agilent RNA 6000 Nano kit and 2100 Bioanalyzer (Agilent, Waldbronn, Germany). Samples with RNA integrity numbers (RIN) >8 [12 (link)] were used to generate a DNA library using a TruSeq Stranded mRNA Sample Preparation kit in accordance with the manufacture’s protocol (Illumina, Berlin, Germany). Essentially, polyadenylated mRNA molecules were enriched from 2 µg of total RNA using poly-T oligo-coated magnetic beads and chemically fragmented under elevated temperature conditions. The fragmented RNA was then reverse-transcribed into cDNA using random hexamers and Superscript II reverse transcriptase and ligated with TruSeq RNA adapters containing a unique DNA sequencing index to enable multiplexing. The DNA libraries were quality-control assessed for fragment-size distribution using an Agilent Technologies 2100 Bioanalyzer and Agilent DNA-1000 Chip kit.