Superscript 3 or 4 reverse transcriptase

Total RNA was isolated using Trizol Reagent (Invitrogen) and treated with DNase I to remove the genomic DNA. cDNAs were synthesized from 1 μg of total RNA using random primer and SuperScript III or IV reverse transcriptase according to the manufacturer’s instructions (Invitrogen). mRNA expression was analyzed by real-time PCR using FastStart Universal SYBR Green Master (Roche Diagnostics, Basel, Switzerland) and the StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Primers used in this study are shown in S1 Table.

Total RNA was extracted using the RNeasy kit (Qiagen) and performing an additional DNase digestion step with the RNase-free DNase set (Qiagen). One µg of RNA was reverse-transcribed into cDNA using superscript III or IV Reverse Transcriptase (Invitrogen). qPCR reactions were carried out on an ABI 7500 and results were normalized according to GAPDH housekeeping gene quantification. Specific primers were predesigned oligos (used 4 nM/well, from primer stock of 100 µM) purchased from Sigma (KiCqStart). qPCR statistical analysis was done using R. Primer sequences used in the study are shown in Supplementary Table 4. Yeast RT-qPCR, including primers used, was performed as previously described [56 ].

Total RNAs were extracted using Direct-zol miniprep RNA kits with TRIzol reagent (Zymo Research). First-strand cDNA was synthesized using Superscript III or IV Reverse Transcriptase (Invitrogen) using a standard protocol provided by manufacturer. Real-time quantitative PCR (qPCR) was performed in Rotor-Gene 6000 (Corbett Research) or QuantStudio 5 384 Optical well plate system (Applied Biosystem) in a standard 10 μl with the 2X SYBR FAST qPCR Master Mix (KAPA Biosystems) and gene specific primers using a standard amplification protocol followed by melt curve analysis. The gene specific DNA primer pairs were designed to cover all the transcripts available currently using GETprime or NCBI primer blast tool, ordered from Sigma-Aldrich and validated on whole brain homogenates (Table S2). The criteria for positive detection of a signal were presence of single peak at specific temperature in the melt curve and presence of a single band in agarose gel electrophoresis. The relative expression levels (2^−ΔCt) were calculated for each target relative to a normalization factor -geometric mean of reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Actin-β or TATA-binding protein (TBP) and importin 8 (IPO8).