Roti quick kit

Liquid nitrogen-frozen tissue was homogenized using a micro-dismembrator (Sartorius, Göttingen, Germany). RNA extraction from mouse tissue was performed using the Roti Quick kit (Carl Roth, Karlsruhe, Germany) followed by RNA purification with the peqGold RNA isolation kit (Peqlab, Erlangen, Germany), according to the manufacturers’ instructions. For RNA quality assessment, RNA integrity numbers (RIN) were measured via Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and samples with RIN number above 6.5 were used for sequencing. RNA library preparation was done using the Illumina TruSeq RNA Sample Prep kit v2 following the manufacturers’ protocol. RNA sequencing was performed as 100-bp paired-end runs on a HiSeq2500 (Illumina). Pools of 16 indexed libraries were sequenced on four lanes. Image analysis and base calling were performed using Illumina Real Time Analysis. CASAVA 1.8 was used for demultiplexing.

Total RNA was prepared from cells using the Roti-Quick-Kit according to manufacturer’s recommendations (Carl Roth) and transcription of 500 ng was performed using random hexamers (Life Technologies) and M-MLV reverse transcriptase (Sigma). For quantitative PCR reactions, we used the SYBR Green LC480 System (Roche). mRNA expression was normalized to the hypoxanthine phosphoribosyl transferase (Hprt) gene as a housekeeping gene (primer sequences available on request) (dCT) and further normalized to the mean value of the control group (ddCT).