BioProject

Purpose: To gain additional insights to candidate factors promoting longevity in Caloric Restricted Conditioned Media (CRCM), we performed transcriptomics analysis (RNA-seq) on BY4741 yeast cells grown in non-restricted SC media supplemented with CRCM, Non-restricted Conditioned Media (NRCM), or water as a control with a goal of identifying physiological responses that could be linked to specific metabolites. Methods: RNA profiles of NR (SC, 2% glucose) treated cells supplemented with water, 2% CRCM, or 2% NRCM at log phase, 24, and 96 hours post-inoculation. These samples were conducted in triplicate. Following inoculation, we harvested cells at log phase, 24 hrs (late diauxic shift), and 96 hrs (stationary phase).Total RNA was isolated using the hot acid phenol method and then processed into Illumina DNA sequencing libraries. Briefly, total RNA was treated with DNase I for 10 min at 37°C and then measured for concentration and quality with an Agilent Bioanalyzer. PolyA mRNA selection was performed on 5µg of the DNase-treated total RNA with the NEBNext Poly(A) mRNA magnetic isolation module (E7490). DNA sequencing libraries were then generated with the NEBNExt Ultra Directional RNA library Prep kit for Illumina (E7420). Libraries were sequenced on an Illumina NextSeq 500 by the UVA Genome Analysis and Technology Core. Results: Sequencing reads were mapped to the sacCer3 genome using bowtie2 with default settings. We preprocessed sequencing data from the UVA genomics core and analyzed differential gene expression in R using DESeq2. We found that In log phase cells, there were no significantly upregulated or downregulated genes in the CRCM or NRCM samples as compared to the water supplemented control (FDR <0.05). At the 24 hr timepoint, CRCM samples diverged from the NRCM and water supplemented control samples, and showed many more differentially regulated genes than the NRCM-treated samples. At the 96 hr timepoint, NRCM samples also became more clearly separated from the control in a PCA plot , though there were still a large number of genes exclusively altered in the CRCM samples. Furthermore, the YCL064C (CHA1) gene clearly stood out as being the most significantly upregulated in conditioned media supplementation. Conclusions: RNAseq data led us to explore YCL064C (CHA1) gene clearly stood out as being the most significantly upregulated in the conditioned media experiments. CHA1 encodes a predominantly mitochondrial L-serine (L-threonine) deaminase that catabolizes these amino acids. RNA-seq based transcriptome analysis allowed us to explore L-serine contribution to longevity phenotype we observed with CRCM. Overall design: RNA profiles of log phase, 24, and 96 hour BY4741 yeast cells grown in SC and supplemented with CR conditioned media, NR conditioned media, or water.