Quantitative Real-time PCR Analyses

Total RNA was extracted from tissues and cell cultures using Trizol Reagent (Ambion) and treated with a RQ1 RNase-free DNase (Promega). One µg of RNA was used to generate cDNA with miScript II RT kit (Qiagen) for snRNA analyses and Superscript III (Invitrogen) for the other genes. Quantitative real-time PCR was performed in triplicate using the primers listed in Supplemental Table 2 with SYBR Green ROX mix (Thermo Scientific) on either Applied Biosystems 7500 fast system or BioRad CFX384. The normalized expression levels were calculated according to the ΔΔCt method. The snRNA, 5 S, 5.8 S, Rpl13a, Ppia, Gapdh, Myh4 and Z⁺Agrn primers and their analyses have been previously described^{43 (link)}. Additional primers have been designed using the free primer design tools from eurofins (eurofinsgenomics.eu) and validated according to the MIQE guidelines.