Fastgene scriptase 2

The experiments were essentially performed as described earlier (28 (link)). Cells were grown to mid log-phase (2 × 10⁷ cells/ml), harvested by centrifugation for 5 min at 4000 rpm, were washed once with 1 ml YPD/1 M Sorbitol/2 mM DTT and resuspended in YPD/1 M Sorbitol/1 mM DTT. Cells were spheroblasted using 1 mg zymolyase (100 mg/ml) and after that diluted in 50 ml YPD/1 M Sorbitol for 30 min at 25°C for recovery. Subsequently, the cells were shifted to 37°C for 1 h. Cells were placed on ice, centrifuged at 2000 rpm for 10min and the pelleted cells were resuspended in 500 μl Ficoll buffer (18% Ficoll 400, 10 mM HEPES pH 6.0) and 1 μl Ribolock. Cells were lysed by addition of 1ml buffer A (50 mM NaCl, 1 mM MgCl₂, 10 mM HEPES pH 6.0). The suspension was mixed and centrifuged at 4000 rpm for 15 min. The supernatant was used for cytoplasmic analyses. RNA was isolated using the Nucleo-Spin RNA Kit (Macherey and Nagel) and reverse transcribed with FastGene Scriptase II (Nippon Genetics) for subsequent qPCR analyses. All values were normalized to the amount of the 21S rRNA. To verify no nuclear contamination in the cytoplasmic fraction, aliquots of the samples were analyzed in western blots for the presence of the cytoplasmic Zwf1 protein and the absence of the nuclear Nop1 protein.

Total RNA was isolated from rat brains using a FastGene™ RNA Premium Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan). Complementary DNA (cDNA) was synthetized using FastGene™ Scriptase II
(NIPPON Genetics Co., Ltd.). Quantitative reverse transcription PCR was performed using a Thermal Cycler Dice Real Time system (Takara Bio Inc., Shiga, Japan) with a KAPA SYBR Fast qPCR Kit
(Roche Diagnostics K.K., Tokyo, Japan). The primer sequences for quantifying cDNA were as follows: 5′-ATACCAGCGGAAACAAGGATTTCA-3′ and 5′-CAGGAATCGGAGTCTGTCAAGTCA-3′ for
Zeb2, and 5′-GTTGTCATCTCAACAGCCAAGCA-3′ and 5′-CAAGTCTTTGGGACAGAGTGGGTAA-3′ for Gtdc1. By monitoring the amplification curves of a test sample and reference
samples that contained between 10¹ and 10⁶ molecules of the mRNA of interest, the number of target mRNA molecules in the test sample was determined and normalized to
the amount of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA. The primer sequences of Gapdh were as follows: 5′-GGCACAGTCAAGGCTGAGAATG-3′and
5′-ATGGTGGTGAAGACGCCAGTA-3′.

Total RNA isolation was carried out with the NucleoSpin RNA Kit from Macherey-Nagel. All steps were conducted according to the manufacturer's description. An additional DNA digestion step was carried out after elution of the RNA. For this, the eluted RNA was mixed with a 10th volume of the reaction buffer and 1 μl DNaseI (Qiagen), according to the manufacturer's description. The digest was incubated for 10 min at 37°C. Afterwards, the RNA was precipitated through sodium acetate ethanol precipitation. For this 0.1 volume 3 M sodium acetate, pH 5.2, 2.5 volumes of 99% pure ethanol and 1 μl Glycoblue were added and incubated over night at –20°C. The collected RNA pellets after centrifugation were resuspended in RNase-free H₂O. The purified RNA was measured via Nanodrop. For cytoplasmic fractionation and Xrn1 digestion experiments, a defined amount of RNA was reverse transcribed with FastGene Scriptase II (Nippon Genetics) for subsequent qPCR analyses.