GSM8532415: rtt105 tel1 rep2; Saccharomyces cerevisiae; OTHER
All colonies from YEP-Galactose plates were collected, pooled together and mixed vigorously. ~80 μl cells were spun down and the genomic DNA was extracted using standard glass beads and phenol chloroform. The genomic DNA was treated with RNase A overnight. The genomic DNA was dissolved with water and adjusted to a concentration of 10 ng/μl. The construction of the amplicon sequencing library was adapted from Illumina's protocol #15044223 Rev. B. Briefly, the sequencing libraries were constructed with two rounds of PCR using KAPA HiFi HotStart ReadyMix polymerase (Roche, 7958927001). For the first round of PCR, 3.2 μL genomic DNA and 0.5 μL 10 μM primers were used for a total volume of 25 μL. The forward and reverse primers targeting the MATa locus are located 11 bp upstream or 18 bp downstream of the HO cleavage site respectively, containing Illumina adapter sequence and 3 bases unique home index as described (7). The thermocycling conditions were: 95 °C for 5 min; 22 cycles of 98 °C for 20 s, 65 °C for 30 s, 72 °C for 3 min; 72 °C for 10 min. After the first round of PCR, 18μL PCR product was purified with an equal volume of AMPure XP beads (Beckman Coulter, A63880). The DNA was eluted with 52.5 μL of 10 mM Tris (pH 8.5) and used as the templates for the second round of PCR. For the second round of PCR, 5 μl template DNA, 5 μL index primer N7xx and 5 μL index primer S5xx from Nextera XT V2 Index kit (Illumina, FC-131-2001) were used for the total volume of 50 μL. The following conditions were used for the second round of PCR: 95 °C for 5 min; 8 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min; 72 °C for 10 min. Then, the PCR product was purified with 1.2x volume of AMPure XP beads and eluted with 10 mM Tris pH 8.5. The average size of each sample was measured with TapeStation and the DNA concentration was measured with Qubit dsDNA BR Assay Kit (ThermoFisher, Q32850). Then, the DNA was diluted to a concentration of 4 nM. An equal volume of DNA from ~20 samples was pooled into the library. The 0.2 M NaOH was used to denature the pooled library and PhiX library (Illumina, FC‐110‐3001). After diluting the libraries with pre-chilled HT1 to 12 pM, pooled library and PhiX library were mixed with the ratio 9:1, further denatured by incubating at 96 °C for 2 min and immediately put in an ice-water bath for 5 min. The denatured combined library was loaded into the MiSeq Reagent Kit v3 (600 cycle) (Illumina, MS-102-3003) for amplicon sequencing.