RNA-Seq of Candida albicans: treated with 1 g/mL OTB
Cultures of C. albicans strain SN152 were inoculated from a fresh colony and grown overnight in YPD at 30C. Cultures were then diluted to an OD595 of 0.1 in 100mL of fresh YPD and treated with 1 g/mL of OTB or an equal volume of DMSO for 4 hours. Cells were then centrifuged for 2min at 5,000rpm, the supernatants were removed, and the samples were quick-frozen and stored at 80C. The Yeast RNA Extraction Kit (Zymo Research, California, America) was used for total RNA extraction. RNA quality and integrity were checked using an Agilent 2100 bioanalyzer. Poly(A)-containing mRNA was isolated from the total RNA by poly(T) oligonucleotide-attached magnetic beads. The DNA probe was used to hybridize rRNA, RNase H selectively digested the DNA/RNA hybridization chain, and DNase I digested the DNA probe. After purification, the required RNA was obtained. The obtained RNA was fragmented and reverse transcribed by the random N6 primers, and then the cDNA double strand was synthesized to form double-stranded DNA. The end of the synthesized double-stranded DNA was flattened and phosphorylated at the 5 end; the 3 end formed a sticky end with a protruding A and then connected to a bubbly joint with a protruding T at the 3 end. PCR amplified the ligation products with specific primers. The PCR product was thermally denatured into a single strand, and then a bridge primer was used to cyclize the single-strand DNA to obtain a single-strand circular DNA library and then sequenced (BGI Genomics, China). After obtaining the raw data by sequencing, we trimmed reads with low quality (a base with a mass value of fewer than 15 accounts for more than 20% of the total base of the read), joint contamination, and high content of N (>5%) of an unknown base to obtain clean reads by the software SOAPnuke (BGI Genomics, China). The analysis of the differential expressed genes was performed by HISAT2 software (v2.0.4) between the OTB treatment and the control(Kim, Langmead et al. 2015), and log2 fold change > 1 and p-value < 0.05 were used as conditions for screening DEGs. Based on the GO and KEGG pathways, functional annotation was performed using the KEGG (http://www.genome.ad.jp/kegg/, accessed on 14 December 2022) databases.