title: scRNAseq in quiescent and refed quiescent cells in Saccharomyces cerevisiae
design: scRNAseq in quiescent and refed quiescent cells in Saccharomyces cerevisiae
library_construction_protocol: Quiescent cells were harvested from a 6 days stationary phase culture following gradient-sorting purification as described in Allen et al. 2006 (10.1083/jcb.200604072). Then, cells were refed with either rich glucose medium (YPD) or non-fermentable medium (YP,lactate 0.5%, ethanol 2%). For cell harvesting and counting, we modified the 10X protocol for methanol fixation (available upon request from 10X genomics). Initially, cell quantity was estimated from the cell suspension concentration in OD600/mL. After fixation, flow cytometry was used to determine cell concentration. to harvest 10000 cells, a 0.2 OD600 equivalent (e.g., 200 μL of cell suspension at 1 OD/mL) of gradient- enriched quiescent cells, or quiescent cells refed for 30 (glucose or lactate ethanol), 60 (glucose or lactate ethanol), 240 (glucose) or 420 minutes (lactate ethanol) was collected in a 2 mL Eppendorf tube, centrifuged for 4 minutes at 800g, and the supernatant was removed. Then, 1.6 mL of chilled 80% methanol was added dropwise to the pellet, incubated at -20°C for 30 minutes, and stored at -20°C or processed further. For rehydration and counting, methanol-fixed cells were brought to 4°C, centrifuged for 4 minutes at 800g, and the supernatant was removed, leaving about 1 μL to evaporate. The pellet was resuspended in 1 mL of wash-resuspension buffer (1 mM DTT, 200 U/mL RNase inhibitor (RiboSafe RNase Inhibitor BIO-65028, Meridian Bioscience), 0.04% BSA, diluted with PBS). The sample was divided into two portions: S1 was stored at 4°C, and S2 was used to assess cell concentration using the Attune NxT Flow Cytometer. The cell suspension volume calculator table was referenced to adjust the dilution to achieve 700-1200 cells/μL. Dilutions were made from S1 before proceeding with step 1.2b of the 10X single-cell protocol. As a  control for the single cell aspect of the experiment, samples taken at the time of the first cell division during release (R3: 240 min for glucose, 420 min for lactate ethanol) were diluted to appropriate concentrations and then mixed 50%-50%. For cell encapsulation, steps 1.2d-e of the 10X Genomics single-cell protocol version v3.1 were modified as follows: a gel beads and Zymolyase suspension was prepared. A 100X stock of Zymolyase (100 mg/mL, MP Biomedicals, 8320932) was added to the gel beads suspension to reach a final concentration of 1.7X before loading on the chip. Previous studies in growing cells used a 1X concentration, but this was not sufficient for quiescent cells. Then, 50 μL of the gel beads and Zymolyase suspension was dispensed into row 2 of the chip, as indicated, before proceeding with library preparation according to the protocol. Library was prepared as indicated for 10X 3'(v3) 