Random hexamer primers

The RNA-seq transcriptome library was prepared following the TruSeq RNA (Illumina, San Diego, CA) sample preparation instructions using 5 μg total RNA. Briefly, messenger RNA was isolated according to the polyA selection method by oligo beads [75 (link)] and then segmented (100 bp to 400 bp) by a fragmentation buffer. Next, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with random hexamer primers (Illumina). Then, the synthesized cDNA was subjected to end repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. The libraries were size-selected for cDNA target fragments of 200–300 bp on 2% low-range ultra-agarose followed by PCR amplification using Phusion DNA polymerase (New England Biolabs) for 15 PCR cycles. After quantification by a TBS-380 fluorometer, the paired-end RNA-seq library was sequenced with the Illumina HiSeq 2000 system (2 × 100 bp read length). Raw reads were archived at the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the accession No. SRP048814.

A transcriptome sequencing (RNA-seq) transcriptome library was prepared with a TruSeq RNA sample preparation kit from Illumina (San Diego, CA) using 1 μg of total RNA. Briefly, mRNA was isolated according to the poly(A) selection method with oligo(dT) beads and then fragmented by fragmentation buffer. Next, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA the was subjected to end repair, phosphorylation, and “A” base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra agarose, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, the paired-end RNA-seq library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150-bp read length). The raw paired-end reads were trimmed and quality controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. Clean reads (see Table S2 in the supplemental material) then were aligned to the reference genome of Candida glabrata CBS 138 (https://www.ncbi.nlm.nih.gov/genome/192?genome_assembly_id=28426). The differential expression analysis was performed using the DESeq software.

Total RNA was isolated using TRIzol® Reagent according to the manufacturer’s protocol (Invitrogen, USA), and genomic DNA was removed using DNase I (Takara, Japan). RNA concentrations, purity and integrity were determined with a NanoDrop ND-2000 (NanoDrop Technologies, Inc., Wilmington, DE, USA) and with an RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). An RNA-seq transcriptome library was prepared using a TruSeq™ RNA sample preparation kit (Illumina, San Diego, CA, USA). Briefly, mRNA was isolated according to the polyA selection method with oligo (dT) beads and then fragmented with fragmentation buffer. Double-stranded cDNA was synthesized using a SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, CA, USA) with random hexamer primers (Illumina, San Diego, CA, USA). Then, the synthesized cDNA was subjected to end repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. The libraries were size-selected for cDNA target fragments of 200–300 bp on 2% Low Range Ultra Agarose and then subjected to PCR amplification using Phusion DNA polymerase (NEB, China) for 15 PCR cycles. After quantification with a TBS-380 instrument, the paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq X Ten (2 × 150 bp read length).

For each of the samples within each treatment per time point, we subjected 5 μg of total RNA to one round of poly A selection on oligo(dT) Serabeads (Illumina mRNA-Seq Kits Cat no. RS-00-0801). The resultant mRNA was fragmented to an average size of 500 bp using divalent cations at 95°C for 5 min prepared following the manufacturer’s recommended protocol (Illumina mRNA-Seq Kits Cat no. RS-100-0801). First strand cDNA synthesis was carried out using Superscript III reverse transcriptase (Invitrogen) and 3 μg random hexamer primers (Illumina) per sample as per the manufactures’ instruction. Second strand cDNA synthesis and RNA-Seq samples were prepared according to the manufacturer’s recommended protocol (Illumina). The fragment size and concentration of resultant libraries were assessed on a Qubit Fluorometer (Invitrogen QuantRNA) and on a Bioanalyzer High Sensitivity Chip (Invitrogen QuantRNA). All libraries were sequenced on Hiseq2500 with 125 bp paired-end reads. The dataset has been deposited in the National Center for Biotechnology Information (NCBI; project accession number PRJNA 393467).

Construction of the four libraries and the RNA-Seq were performed by the Biomarker Biotechnology Corporation (Beijing, China). Five micrograms of total RNA from each sample (EM, EH, LM, and LH) were used to construct the sequencing libraries. RNA sequencing libraries were generated using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) following the manufacturer’s recommendations. Briefly, the poly (A)-containing mRNA molecules were purified from the total RNA by using the poly-T oligo-attached magnetic beads (Illumina, San Diego, CA, USA). Then, mRNA was broken into short fragments, which were used as templates for cDNA synthesis. Double-stranded cDNA was synthesized using SuperScript II, buffer, dNTPs, RNaseH, DNA polymerase I, and random hexamer primers (Illumina). After that, the ‘A’ tail was added to the 3′ ends of the repaired cDNA fragments and the Illumina’s paired-end adapters were ligated to the cDNA ends. The products from the ligation reaction were amplified by PCR. The PCR productions were purified by magnetic beads (Illumina) and dissolved in EB solution to generate the sequencing libraries. The quantity and quality of each sequencing library were tested by the Agilent 2100 Bioanalyzer. Finally, the four libraries were sequenced separately on Illumina HiSeq™ 2500 platform.

RNA-seq transcriptome libraries were prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA, United States) using 1 μg of total RNA. Firstly, messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer. Secondly, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA, United States) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and “A” base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length). Statistical methods were used to calculate the base distribution and quality fluctuation of each cycle of all sequenced reads, which could intuitively reflect the library construction quality and measurement of sequenced samples from a macro-perspective.