RNA sequencing (RNA-SEQ) of RRD1 knockout yeast strains with Rapamycin treatment

ABSTRACT: Purpose: We develop a multivariate polynomial temporal genetic association (MPTGA) approach for detecting temporal genetic loci of quantitative traits monitored over time in a population. To dissect causal regulators under each temporal genetic locus, we develop a temporal genetic causality test (TGCT) for inferring causal relationships between traits linked to the locus. We applied MPTGA and TGCT to simulated data sets and a yeast population that was molecularly profiled over six time points in response to treatment with the drug rapamycin. We demonstrate significantly increased power to detect genetic loci modulating gene expression traits over time in this population and to resolve the causal regulators of dynamic expression quantitative loci hot spots, thereby increasing the power to elucidate regulatory networks. We experimentally validated a teQTL (temporal expression Quantitative Trait Locus) hot spot locus interacting with rapamycin treatment over time, which could only be identified using our temporal genetic association procedure. We experimentally validated RRD1, a cis regulated gene in this locus, as the causal regulator for this teQTL hot spot. Methods: The wild type strain BY4730 and RRD1 knockout strain YSC6273-201925697 were obtained from Thermo Scientific Open Biosystems. Yeast was grown in YPD medium to log-phase in shaken flasks at 30°C. Total RNA was extracted as described previously. For rapamycin treatment, 100nM rapamycin (Cayman Chemical, Ann Arbor, MI) was added to the medium after yeast grew to log-phase. After culture for 50 minutes, total RNA was extracted the same as above. All experiments were repeated 3 times on three different days. Approximately 250 ng of total RNA per sample were used for library construction by the TruSeq RNA Sample Prep Kit (Illumina) and sequenced using the Illumina HiSeq 2500 instrument with 100nt single read setting according to the manufacturer's instructions. Sequence reads were aligned to yeast genome assembly using Tophat . Total 6,932 yeast transcripts were quantified using Cufflinks, and 5,542 of them overlap with transcripts on Yeast Genome 2.0 Arrays from Affymatrix, which was used for generating the yeast F2 time course data. The 5,542 transcripts were used in further analysis. Differentially expressed genes were defined by CuffDiff. At q-value <0.01, 64 and 581 were in RRD1 ko signature without rapamycin (RRD1 ko no treatment vs. wild type no treatment) and RRD1 ko signature with rapamycin (RRD1 ko with rapamycin vs. wild type with rapamycin), respectively. Conclusions: Integration of temporal and genetic data has the potential to enhance the power to resolve causal relationships and to provide a more accurate view of regulatory networks in complex biological systems.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE86786 | GEO | 2018/12/21

REPOSITORIES: GEO