Rnaeasy purification kit

A total of 10⁸ G1 phase cells were harvested, released into fresh YPD medium,, grown for 10 min prior to harvesting by centrifugation and stored at −80°. Total RNA was extracted using the RNAeasy purification kit (Qiagen) and glass bead lysis in a Biospec Mini 24 bead-beater, as previously described (Sellam et al. 2009 (link)). cDNA was synthesized from 2 µg of total RNA using the SuperScript III Reverse Transcription system [50 mm Tris-HCl, 75 mm KCl, 10 mm dithiothreitol, 3 mm MgCl₂, 400 nm oligo(dT)₁₅, 1 m random octamers, 0.5 mm dNTPs, and 200 U Superscript III reverse transcriptase]. The total volume was adjusted to 20 µl, and the mixture was then incubated for 60 min at 42°. Aliquots of the resulting first-strand cDNA were used for real-time quantitative PCR (qPCR) amplification experiments. qPCR was performed using the iQ 96-well PCR system for 40 amplification cycles and QuantiTect SYBR Green PCR master mix (Qiagen). Transcript levels of RNR1 were estimated using the comparative Ct method, as described by Guillemette et al. (2004) (link), and the C. albicans ACT1 open reading frame as a reference. The primer sequences were as follows: RNR1-forward: 5′-GACTATCTACCATGCTGCTGTTG-3′; RNR1-reverse: 5′-GGTGCAACCAACAAGGAGTT-3′; ACT1-forward: 5′-GAAGCCCAATCC AAAAGA-3′; and ACT1-reverse: 5′-CTTCTGGAGCAACTCTCAATTC-3′.

The extraction and purification of total RNA was performed using the RNAeasy purification Kit (QIAGEN) combined with RNaseFree DNase (QIAGEN) treatment according to the manufacturer’s protocol. RNA concentration and quality was determined using the NanoDrop 2000 spectrophotometer before downstream processing. Quality of the purified RNA was tested on an Agilent 2200 Tapestation using RNA screentape. Libraries were prepared using the TruSeq stranded total RNA with RiboZero kit (Illumina). Quality and quantity of the cDNA libraries were assessed on an Agilent 2200 Tapestation (D1000 screentape). Libraries were run on an Illumina NextSeq500 using the High Output 75 cycles kit (2 × 36 cycles, paired-end reads, single index). Quality checks on the raw RNA-Seq data files were done using FastQC [52 (link)]. Alignment of the RNA-Seq paired-end reads was to the GRCm38 [53 (link)] version of the mouse genome and annotation using HiSat2 [54 (link)] and TopHat [55 (link)]. Gene expression levels were quantified using the feature Counts tool [56 (link)] available on Galaxy.org DESeq2 [57 (link)] was used to generate a list of differentially expressed protein-coding genes between. Calculations were performed with R version 3.3.1 in R-Studio (version 0.99.903). RNA-Seq data is available as GEO accession number GSE190668.