RNAseq and ribosome profiling of yeast cells grown under glucose restriction condition.

We investigate the mechanism by which glucose restriction extends yeast replicative lifespan, using an approach that combines ribosomal profiling and RNA-seq. We systematically compared the translational and transcriptional profiles of cells grown in glucose restriction and normal media, uncovering groups of functionally related genes that are up or down regulated. We used two different protocols to grow cell culture in SD vs. GR conditions. 1) protocol-1: Quick Dilution from SD (2% glucose) to GR (0.05% glucose): the initial cell culture was incubated in SD medium overnight to OD600 0.8~1.0, then diluted by 5 fold using SD media and incubated for another 4 hours. The sample was then divided equally into two aliquots. One was diluted 40 times by pre-warmed SD medium with 0% glucose to reach final 0.05% glucose concentration (the sample), and the other diluted 40 times by pre-warmed SD medium with 2% glucose (the control). Both samples were incubated for another hour before harvesting. All the steps were carried out at 30°C. 2) protocol-2: Spin-down and re-suspension from SD to GR : the initial cell culture was incubated in SD medium overnight to a OD600 0.8~1.0, then diluted by 5 fold using SD medium and incubated for another 4 hours. The sample was then divided equally into 2 aliquots. Cells were separated from the media by spin-down at 2000 rcf for 5 minutes and re-suspended into SD and GR media respectively. All samples were incubated for for another hour before harvesting. All the steps were carried out at 30°C. Ribosomal profiling experiments were carried out using the protocol developed by Ingolia et al. Raw sequences were obtained from Illumina Hiseq 2000.