title: GSM5856778: cardiac tissue, wt, rep 3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from mouse tissues using TRIzol reagent (Life Technologies). Initially, 50-100 mg of fresh or snap-frozen tissue was placed in 2 mL tubes (Peqlab) together with ceramic beads (Mobio, Dianova GmbH) and 1 mL TRIzol. The tissue was homogenized using a Precellys 24 fast-prep machine (Bertin) at 5500 rpm for 2x 20 sec.  Homogenates were then transferred to a new tube and it proceeded with the addition of chloroform and subsequent phase separation according to the manufacturer's instruction. The RNA pellet was resuspended in RNase/DNase free H2O (Gibco). RNA isolation for RNAseq was performed using the 'Direct-zol RNA MiniPrep Kit' (Zymo Research) according to the manufacturer's instruction. Purified RNA was submitted to the Cologne Centre for Genomics for further processing. Libraries were prepared using the 'TruSeq mRNA Stranded Sample Preparation Kit' (Illumina) and validated on a tape station (Agilent). Equimolar amounts of libraries were pooled, quantified (Peqlab KAPA Library Quantification Kit, Applied Biosystems 7900HT Sequence Detection System) and finally sequenced on a 'NovaSeq 6000 sequencer' (Illumina).