Cells were grown in triplicates for each condition in YPD at 25°C until logarithmic phase and heat shocked at 37°C for 30 min in a shaking water bath. Cells were harvested by centrifugation and RNA was extracted using the RiboPure RNA Purification Kit for Yeast (Thermo Fisher Scientific). Libraries were prepared using Illumina TruSeq Stranded mRNA (polyA selection) and quality checked using Quant-iT (DNA BR) and CaliperGX. Minimum concentration was 5 ng/μl and a size of >300 base pairs. RNA-Seq was performed on Illumina HiSeq2500 (2 × 101–base pair paired end read). The sequencing reads were mapped to the sacCer2 genome with ensemble annotation Saccharomyces cerevisiae EF2.62 using Tophat (v 2.0.4). Gene counts and FPKMs were estimated using HTSeq (v 0.6.1) and Cufflinks (v 2.1.1), respectively. Principal component analysis was performed based on gene FPKMs using prcomp() function in R (v 3.5.3). Differential gene expression analysis was performed using DESeq2 (v 1.22.2). The genes with p value < 0.01, q value < 0.05 and absolute log2 fold change >1 were identified as differentially expressed.
Ribopure rna purification kit for yeast
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania
The RiboPure RNA purification kit is designed for the isolation of total RNA from yeast samples. The kit utilizes a phenol-based extraction method to efficiently capture and purify RNA molecules from yeast cells.
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Lab products found in correlation
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Kapa sybr fast universal qpcr kit, by Roche (3 mentions)
Sybr green qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Goscript reverse transcription system, by Promega (2 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
Myiq single color real time pcr detection system, by Bio-Rad (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Truseq stranded mrna, by Illumina (1 mentions)
3 mrna seq library prep kit, by Lexogen (1 mentions)
Ribo zero yeast kit, by Illumina (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Dextrose, by Merck Group (1 mentions)
Peptone, by Merck Group (1 mentions)
Trueseq adapters, by Illumina (1 mentions)
L9650, by Merck Group (1 mentions)
Hiseqv4, by Illumina (1 mentions)
T75 cell culture flask, by Corning (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Maxima sybr green qpcr master mix, by Thermo Fisher Scientific (1 mentions)
14 protocols using ribopure rna purification kit for yeast
1
RNA-Seq Analysis of Heat Stress Response in Yeast
2
Yeast Transcriptome Analysis in SD Media
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Total yeast RNA after 4-h cultivation in synthetic dextrose (SD) or SD media with adenine omitted (SD ade−) was isolated with a RiboPure™ RNA Purification Kit for yeast (Thermo Scientific, Waltham, MA, USA). RNA samples for each condition were harvested in triplicate. Cell pellets from 50 mL suspensions were frozen in liquid N2 and stored at −80 °C. RNA samples were prepared using 3′ mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to the manufacturer’s protocol. Yeast transcriptome was analyzed using MiSeq (Illumina, San Diego, CA, USA) NGS data analysis. Sequencing reads were quality filtered (Q = 30), Illumina adapters and poly-A tails were removed, and reads at least 100 nt in length were selected for further processing using cutadapt (see File S4 for details). S288c reference genome from yeastgenome.org was used to identify gene transcripts.
Genes with lower than 1 count per million (CPM) in fewer than 2 samples were filtered out. The Benjamini and Hochberg method was used to calculate multiple comparison adjusted p-value as false discovery rate (FDR). FDR < 0.001 with logFC > 2 was set as a threshold for significance. Expression data set were submitted to the European Nucleotide Archive (ENA) database, under accession no. PRJEB40525.
Genes with lower than 1 count per million (CPM) in fewer than 2 samples were filtered out. The Benjamini and Hochberg method was used to calculate multiple comparison adjusted p-value as false discovery rate (FDR). FDR < 0.001 with logFC > 2 was set as a threshold for significance. Expression data set were submitted to the European Nucleotide Archive (ENA) database, under accession no. PRJEB40525.
3
Isogrowth RNA Sequencing of S. cerevisiae
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For isogrowth RNA sequencing, S. cerevisiae strain BY4741 was grown in 7 conditions of varying ratios of LiCl (Sigma Aldrich, L9650) and cycloheximide (Sigma Aldrich, 37094), ensuring 50% growth inhibition, and in YPD [yeast extract (Sigma Aldrich cat. No. Y1625) 1% w/v, peptone (Sigma Aldrich cat. No. 91249) 2% w/v, dextrose (Sigma Aldrich cat. No. D9434) 2% w/v] containing no drug (Table 1). The frozen stock was diluted 130-times into drug containing wells and 100-times more into wells without drug. The cells were incubated for ~17 hrs to an absorbance of ~0.1 on the 96-well plate, corresponding to OD 600 ~0.5. Cells were then harvested and the RNA extraction was performed using RiboPure RNA Purification Kit for yeast (Thermo Scientific, AM1926). The extracted RNA was sent to the Next Generation Sequencing Facility of the Vienna Biocenter Core Facilities, where it was rRNA depleted using Illumina Ribozero Yeast Kit, multiplexed using Illumina True Seq adapters, single-end 50bp sequenced using Illumina HiSeqV4 and demultiplexed.
4
Candida albicans Biofilm Modulation
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Overnight cultures were standardised as described above and C. albicans biofilms grown in T-75 cell culture flasks (Corning, USA) for 4 h in 5% CO2. Following incubation, media was removed, and biofilms washed before L. crispatus was added for an additional 2, 4 or 20 h. At each time point the media was removed and biofilms washed with PBS before being scraped in to 1 mL of RNAlater (ThermoScientific, Loughborough, UK). Spent media from biofilms was retained and pH monitored throughout the experiment. RNA was extracted from microbial biofilms using the preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.12.430906 doi: bioRxiv preprint RiboPure™ RNA Purification Kit for yeast (ThermoScientific, Loughborough, UK), following manufacturer's instructions. Integrity of RNA was assessed using a Bioanalyser system and genome-wide Candida transcripts sequenced using the Illumina NOVASeq6000 sequencing platform (Edinburgh Genomics). A summarised illustration of the experimental and bioinformatics pipelines can be found in Supplementary Figure 1.
The copyright holder for this this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.12.430906 doi: bioRxiv preprint RiboPure™ RNA Purification Kit for yeast (ThermoScientific, Loughborough, UK), following manufacturer's instructions. Integrity of RNA was assessed using a Bioanalyser system and genome-wide Candida transcripts sequenced using the Illumina NOVASeq6000 sequencing platform (Edinburgh Genomics). A summarised illustration of the experimental and bioinformatics pipelines can be found in Supplementary Figure 1.
5
Yeast RNA Extraction and qPCR Analysis
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RNA was harvested from yeast cells in exponential growth in Yeast Extract-Peptone-Dextrose (YPD) broth using the RiboPure RNA purification kit for yeast (Invitrogen), and then treated with DNaseI. cDNA was synthesized using Verso cDNA synthesis kit (Fisher Scientific). Genomic DNA contamination was checked by PCR with primers flanking the intron of C. glabrata ACT1. Primers used are summarized in Supplemental Table 1 43 (link),58 ,59 (link). Quantitative PCR with SYBR Green qPCR Master Mix (Fisher Scientific) was performed with 1:10 diluted cDNA. Target gene expression was calculated using the ΔΔCT method, with normalization to the housekeeping genes ACT1 and Cg18S. All experiments were done in independent biological triplicates and are shown as mean with standard deviation (SD) for each time point.
For mitochondrial copy number determination, we used quantitative PCR, with COX1 as target for mitochondrial gene and ACT1 for control (Supplemental Table 1 ). The amplification was performed using Maxima SYBR Green qPCR Master Mix (ThermoFisher Scientific, Waltham, MA, USA).
For mitochondrial copy number determination, we used quantitative PCR, with COX1 as target for mitochondrial gene and ACT1 for control (
6
Yeast RNA Extraction and qPCR Analysis
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RNA was harvested from yeast cells in exponential growth in YPD broth using the RiboPure RNA purification kit for yeast (Invitrogen AM1924), and then treated with DNaseI. cDNA was synthesized using Verso cDNA synthesis kit (Fisher Scientific AB1453A). Genomic DNA contamination was checked by PCR with primers flanking the intron of C. glabrata ACT1. Primers used throughout the study are listed in Supplementary Table S5 46 (link),63 (link),64 (link). Quantitative PCR with SYBR Green qPCR Master Mix (Fisher Scientific FERK0223) was performed with 1:10 diluted cDNA. Target gene expression was calculated using the ΔΔCT method, with normalization to housekeeping genes ACT1 and Cg18S. Data are shown as mean with standard error of mean (SEM) of at least 3 independent experiments.
7
Quantification of Candida Transcripts in Neutrophils
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C. albicans cells were incubated with MPO-/- mouse neutrophils in 8-well μ-Slide ibiTreat chambers as described above. After 90 min incubation, the supernatant was removed, and the remaining cells (neutrophils and ingested Candida) were scraped off the wells in lysis buffer of the RiboPure RNA purification kit for yeast (Ambion, ThermoFisher Scientific). Total RNA was extracted following the manufacturer’s instructions and concentrated using the RNA Clean & Concentrator-5 kit (ZymoResearch). cDNA was synthesized using SuperScript II Reverse Transcriptase (ThermoFisher Scientific) following the manufacturer’s instructions. Transcript quantification was conducted through real-time PCR analysis using SYBR green. The oligos used are listed in S3 Table . The experimentally validated TAF10 transcript [64 (link)] was used to normalize the qPCR data as we have done before [63 (link)].
8
Yeast Transgene Expression Analysis
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Yeast transformants grown as described above were shifted to SGal-Ura for transgene induction. Four hours after the galactose shift, cells were harvested for RNA isolation. RNA was isolated using the RiboPure™ RNA Purification Kit for yeast (Ambion™, Thermo Fisher Scientific) following the manufacturer’s instructions. Approximately 50 ng RNA were used for each RT-PCR, using AccessQuick™ RT-PCR System (Promega Corporation, Madison, USA). The primers used to detect the expression of the transgene were presented in S3 Table . For each transformant, the expression of actin gene (ACT1) was used as control. The RT-PCR cycling conditions were 45°C, 30 min (for reverse transcription) followed by PCR: 95°C for 2 min, and 25 cycles of 95°C for 30 s, 55°C for 30 s, 68°C for 48 s.
9
Mn2+ Stress Response in Yeast
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Wild-type cells BY4743 (TRPY1/TRPY1), heterozygous (trpy1Δ/TRPY1), and homozygous (trpy1Δ/trpy1Δ) diploid cells from overnight pre-cultures were inoculated at OD600 = 0.1 in MM or LMeMM and grown to OD600 = 0.5 before Mn2+ was added to final concentration 10 µM, then incubated for 2 additional hours before being harvested for total ribonucleic acid (RNA) isolation. Total RNA was isolated using the RiboPure™ RNA Purification Kit for yeast (Ambion™, Thermo Fischer Scientific, Vilnius, Lithuania) following the manufacturer’s instructions. Approximately 500 ng of total RNA was transcribed into cDNA using GoScript™ Reverse Transcription System (Promega, Madison, WI, USA). Finally, a total of 10 ng cDNA was used for each qRT-PCR done with the GoTaq® qPCR Master Mix (Promega). Each reaction was performed in triplicate using MyiQ Single-Color Real-Time PCR Detection System (BioRad, Hercules, CA, USA). Expression of TRPY1 mRNA was normalized to the relative expression of ACT1 in each sample. The qRT-PCR cycling conditions were 95 °C for 1 min, and 40 cycles of 95 °C for 10 s, 59 °C for 10 s, 72 °C for 12 s. The primers used for amplification of cDNA were: TRPY1-F: 5′-AGATTCTCAG GGTTACGTTA, TRPY1-R: 5′-CAATATGGAATACCACTCAC; ACT1-F: 5′-GGTTGCTGCTTTGGTTATTG, ACT1-R: 5′-CAATTGGGTAACGTAAAGTC.
10
Quantifying Gene Expression in Yeast
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RNA was extracted from cells grown in YPD using a RiboPure RNA Purification Kit for Yeast (Ambion, Invitrogen). cDNA was synthesized from DNase I-treated RNA using Superscript III Reverse Transcriptase (Invitrogen) and qPCR was performed using KAPA SYBR Fast Universal qPCR Kit (KAPA Biosystems) with primers listed Table 3 . Quantification was performed using the 2–ΔΔCT method and expression was normalized to TAF10 (Livak and Schmittgen, 2001 (link); Teste et al., 2009 (link)).
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