Illumina HiSeq 4000 sequencing; RNA-Seq in midbrain of Coq9R239X mice treated with Rapamycin
RNA-Seq in midbrain of Coq9R239X mice treated with Rapamycin
For the RNA-Seq we dissected the midbrain of the animals. The tissue samples were rapidly frozen in liquid nitrogen and kept them at -80ºC  RNA was extracted following the TRI Reagent Solution protocol from Applied Biosystems Library preparation and sequencing was performed at the Transcriptome and Genome Analysis Laboratory, NGS Core Unit at the Universitzy Medical Center Göttingen. Quality and integrity of RNA was assessed with the Fragment Analyzer from Advanced Analytical by using the standard sensitivity RNA Analysis Kit (DNF-471). All samples selected for sequencing exhibited an RNA integrity number over 8. RNA-seq libraries were performed using 500 ng total RNA of a non stranded RNA Seq, massively-parallel mRNA sequencing approach from Illumina (TruSeq RNA Library Preparation Kit v2, Set A; 48 samples, 12 indexes, Cat. N°RS-122-2001). For accurate quantitation of cDNA libraries a fluorometric based system, the QuantiFluor™dsDNA System from Promega were used. The size of final cDNA libraries was determined by using the dsDNA 905 Reagent Kit (Fragment Analyzer from Advanced Bioanalytical) exhibiting a sizing of 300 bp in average. Libraries were pooled and sequenced on the Illumina HiSeq 4000 (SE; 1 x 50 bp; 30-35 Mio reads/sample).
Experimental Factor: compound
vehicle
Experimental Factor: genotype
Coq9+/+