title: Illumina Genome Analyzer IIx sequencing; Plasticity of Mitochondrial DNA Inheritance and its Impact on Nuclear Gene Transcription in Yeast Hybrids grown on YP Glycerol 28C
design: Plasticity of Mitochondrial DNA Inheritance and its Impact on Nuclear Gene Transcription in Yeast Hybrids grown on YP Glycerol 28C
library_construction_protocol: To isolate RNA, cells were pelleted, flash frozen using liquid nitrogen and ground to a fine powder and pre-chilled to −80 °C. The experiment had four genotypes (1. Saccharomyces cerevisiae BY4741, 2. Saccharomyces uvarum NCYC 2669, 3. hybrid BY4741 x NCYC 2669 with S. cerevisiae mitochondria, 4. hybrid BY4741 x NCYC 2669 with S. uvarum mitochondria), grown at 28 degrees, on YP glycerol. Yeast strains and media. Saccharomyces cerevisiae BY4741 were obtained from Thermo Scientific, UK. A KanMX cassette was previously engineered into the neutral AAD3 locus of S. cerevisiae BY4741 (MATa his3∆0 leu2∆0 met15∆0 ura3∆0) to confer selectivity (Piatkowska EM, Naseeb S, Knight D, Delneri D. 2013. Chimeric protein complexes in hybrid species generate novel phenotypes. PLoS Genet. 9:e1003836.). Saccharomyces uvarum NCYC 2669 was obtained from the National Collection of Yeast Cultures, UK. Yeast strains were maintained on YPDA (2 % (w/v) Bacto Peptone, 1 % (w/v) Bacto Yeast Extract, 2 % (w/v) glucose, and 2 % (w/v) agar). Hybrid growth was assessed on either YPDA or YP + glycerol (2 % (w/v) Bacto Peptone, 1 % (w/v) Bacto Yeast Extract, 2 % (w/v) glycerol, and 2 % (w/v) agar).Construction of hybridsS. uvarum NCYC 2669 was sporulated by inoculating into pre-sporulation media (0.8 % (w/v) Bacto Yeast Extract, 0.3 % (w/v) Bacto Peptone, and 40 % glucose) overnight, pelleting the cells and spreading thinly on minimal sporulation plates (1 % (w/v) potassium acetate and 2 % agar) and then incubating for five days at 16 °C, or until tetrads were observed microscopically. Tetrads were collected and incubated in 40 µL 1.5 M sorbitol at 37 °C for 7 minutes with 0.2 mg/mL Zymolyase (MP Biomedicals, USA) to disrupt the integrity of asci and release haploid spores. Individual S. uvarum NCYC 2669 mixed mating type spores and S. cerevisiae BY4741 MATa haploids were placed into physical contact for mating using a Singer Instruments MSM micromanipulator on a surface of either YPD agar or YP + glycerol agar. Plates were incubated at 10 °C, 16 °C, or 28 °C until colonies of 2-3 mm were visible. Colonies were then replica plated onto SDA + G418 selective plates (0.17 % (w/v) YNB without additional amino acids, 0.5 % (w/v) ammonium sulphate, 2 % (w/v) glucose, 2 % (w/v) agar, and 300 µg/mL G418 geneticin). These conditions exclusively select for hybrids, which can both tolerate geneticin and grow in the absence of additional amino acids. Plates were re-incubated until hybrid colonies were visible. The colonies were then re-streaked onto SDA + G418 selective media before testing. The hybrid strains passed through roughly 50 generations. Strains were stored at −80 °C in 15 % (v/v) glycerol. The experiment had four genotypes (1. Saccharomyces cerevisiae BY4741, 2. Saccharomyces uvarum NCYC 2669, 3. hybrid BY4741 x NCYC 2669 with S. cerevisiae mitochondria, 4. hybrid BY4741 x NCYC 2669 with S. uvarum mitochondria), grown at 28 degrees, on YP glycerol. Total RNA was extracted from each of three independently constructed hybrids of BY4741 × NCYC 2669 with S. cerevisiae type mtDNA and three independently constructed hybrids of BY4741 × NCYC 2669 with S. uvarum mitochondria that had been constructed at 28 °C on YPD. Cultures were inoculated in either 5 mL of YP + glycerol or YPD and grown overnight at 28 degrees, 200 rpm. Cultures were scaled up in 50 mL fresh media at the appropriate temperature, 200 rpm to obtain mid log phase of between OD600 0.5 and OD600 0.8, at which cells were actively and rapidly replicating. To isolate RNA, cells were pelleted, flash frozen using liquid nitrogen and ground to a fine powder and pre-chilled to −80 °C. To break open the cell walls, Trizol (Ambion, Life Technologies, USA) was dispensed drop-wise onto the frozen pellet, which was then left to defrost. The thawed mixture was left for 5 minutes to allow dissociation of the nucleoprotein complexes. The mixture was pelleted for 10 minutes at 12,000 × g and 200 µL chloroform was added to every 1 mL of clear supernatant. Mixture was inverted and left for 10 minutes at room temperature. Phases were separated by centrifugation at 12,000 × g for 5 minutes. The upper clear aqueous RNA phase was transferred to fresh tubes and the RNA was precipitated with 1:1 isopropanol left for 10 minutes at room temperature. The mixture was centrifuged at 12,000 × g for 10 minutes and the supernatant was removed. The RNA pellet was washed with 1 mL (DEPC)-treated 70 % ethanol, air dried, dissolved in 1:1 lithium chloride (Ambion, Life Technologies, USA): DEPC-H2O and incubated overnight at −20 °C to precipitate a refined RNA pellet. The pellet was washed twice with DEPC-treated 70 % ethanol as before and then redissolved in 50 µL DEPC-H2O. The integrity of the RNA was checked on 1.5 % agarose gel. The quantity and quality of RNA was assessed using a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA). Truseq stranded mRNA Assay.