GSM8478316: nuc1 spt3, DSB, Stationary 8day, biol 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 sequencing library was adapted from Illumina's amplicon sequencing protocol #15044223 Rev. B. Two rounds of PCR were performed to construct the sequencing library. For each sample, 3.2 μl genomic DNA, 0.5 μl 10 μM primers and 12.5 μl KAPA HiFi HotStart ReadyMix (Roche, 7958927001) was used for the first round of PCR for a total volume of 25 μl. The forward primers are 66-mers that contain (5' to 3'): 33 bases Illumina adapter sequence, 3 bases unique home index, and 30 bases targeting the Mata locus which is located 11 bp upstream the HO cleavage site. The reverse primers are 66-mers that contain (5' to 3'): 34 bases Illumina adapter sequence, 3 bases unique home index, and 29 bases targeting the MATa locus which is located 18 bp downstream the HO cleavage site. The following conditions were used for the first round of PCR: 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. 18μl PCR product was purified with 20μl AMPure XP beads (Beckman Coulter, A63880) and eluted with 52.5 μl 10 mM Tris pH 8.5. For the second round of PCR, 5 μl purified PCR product, 5 μl Nextera XT V2 Index (Illumina, FC-131-2001) primer N7xx, 5 μl index primer S5xx, and 25 μl KAPA HiFi HotStart ReadyMix were used. The total volume of PCR is 50 μl. The following conditions were used for 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. 40 μl PCR product was purified with 48 μl AMPure XP beads and eluted with 27.5 μl 10 mM Tris pH 8.5. The DNA concentration of each sample was determined with Qubit dsDNA BR Assay Kit (ThermoFisher, Q32850) and the average size of the DNA was determined with TapeStation. The DNA was diluted to 4 nM using 10 mM Tris pH 8.5. An equal amount of DNA was pooled from ~20 samples into the library. The pooled library and PhiX library (Illumina, FC‐110‐3001) were denatured with 0.2 M NaOH separately and diluted with pre-chilled HT1 to 12 pM. 540μl pooled library and 60 μl PhiX were mixed, incubated at 96 °C for 2 min and immediately placed 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.