Illumina HiSeq 2000 paired end sequencing; Mapping mitochondrial DNA recombination in yeast
Mapping mitochondrial DNA recombination in yeast
The two parental haploid strains were grown overnight in 5 ml YPD (Yeast Extract 10 g/L, Bactopeptone 20 g/L, Dextrose 20 g/L). 100 microliter of each cell suspensions were plated on YPD agar. The cells were incubated at 30C for 24h to allow them to mate. The cell layer was then scraped from the plate and resuspended in 1 ml sterile water. 200 microliter of this suspension were plated on selective media to allow for the selection of diploid cells. This step was repeated three times per cross. The cells were grown for 48h at 30C. The mitochondrial DNA extraction protocol was adapted from Defontaine et al. (1991). The cells were scraped from the plates and resuspended in 1 ml water. They were harvested by centrifugation 3 min at 6000 rpm and washed once with 1 ml water. The cell pellet was washed once in 1ml water and once in 1ml Wash Buffer (1.2 M Sorbitol, 50 mM Tris pH 7.5, 50 mM EDTA, 2% beta-mercaptoethanol). The pellet was then resuspended in 1 ml Solution A (0.5 M Sorbitol, 50 mM Tris pH 7.5, 10 mM EDTA, 2% beta-mercaptoethanol), 100 U Zymolyase 100T (Amsbio) were added and the cells were incubated at 37C for 45 min under gentle horizontal shaking (400 rpm). The suspension was then centrifuged at 4000 rpm for 10 min. The supernatant was transferred to a new 1.5 ml tube and centrifuged at 14000 rpm for 15 min. The crude mitochondrial pellet was washed three times with 1 ml Solution A, resuspended in 400 microl Solution B (100 mM NaCl, 10 mM EDTA, 50 mM Tris pH 8) and incubated at room temperature for 30 min to allow for lysis. The mtDNA was then purified by phenol-chloroform extraction followed by ethanol precipitation. The mtDNA pellet was resuspended in 50 microl EB buffer (10 mM Tris pH 8). RNAs were degraded by RNAse cocktail treatment for 1h at 37C. The mitochondrial DNA libraries were generated using a protocol adapted from Wilkening et al. (2013). 3 to 10 micrograms of mitochondrial DNA were sheared using the S220 Focused-ultrasonicator (Covaris) with the following conditions: Duty cycle: 20%, Intensity: 5, Cycles/burst: 200, Duration: 45 sec, Temperature: 4C. The 350 bp fragments were purified using 1 volume Agencourt AMPure XP beads (Beckman Coulter). We performed end-repair, A-addition and adapter ligation on these fragments; heat inactivation of the enzymes was performed after each step. The samples were purified using 1 volume Agencourt AMPure XP beads and the libraries were amplified by PCR (98C for 45 sec, 12 cycles: (98C for 15 sec, 65C for 30 sec, 72C for 30 sec), 72C for 5 min). This step was performed using 2 to 10 ng of DNA, 2X Phusion High Fidelity PCR Master Mix (NEB) and 0.2 microM PE1.0 and PE2.0 primers (Illumina). The PCR products were then cleaned with 1 volume Agencourt AMPure XP beads and the yield was quantified with a Qubit Fluorometer (Life Technologies). Up to 13 samples were pooled and purified for 400-450 bp fragments on an E-gel (Life Technologies). The multiplexed libraries were sequenced on a Hiseq 2000, yielding between 2.8 and 42 million reads per sample
Experimental Factor: genotype
MATa/MATalpha hodelta/ho::hisG LYS2/lys2delta GAL2/gal2delta ura3delta/URA3 his3delta/HIS3  ntg1delta::KanMX-klURA3/ntg1delta::KanMX-klURA3
Experimental Factor: strain
Hybrid S288c YJM789