GSM8023804: BLX assay timepoint 3 (36h with geneticin); Saccharomyces cerevisiae; OTHER
Plasmid isolation was achieved using the protocol described by Fowler et al. (2014), which in summary used a freeze-thaw cycle and zymolyase cell wall digestion steps were coupled with Qiaprep spin miniprep kit (Qiagen) column purification Samples from each timepoint were prepared for next-generation sequencing using a single limited-cycle PCR. Barcodes were amplified using primers possessing overhangs that contained sequencing primer annealing sequences, unique dual indexes, and P5/P7 Illumina adapter sequences (Table S1). 50 µl PCR reactions were performed for each timepoint using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) with 20 to 50ng of template isolated plasmid DNA. Each reaction was then thermocycled using the following conditions: (1) initial denaturation at 98°C for 30 seconds, (2) incubation at 98°C for 8 seconds, (3) incubation at 69°C for 20 seconds, (4) incubation at 72°C for 8 seconds, (5) incubation at 72°C for 7 minutes. Steps 2 to 4 were repeated for 12 to 19 cycles (< 20). Multiple replicate 50 µl reactions were performed for each timepoint to ensure enough PCR product was generated. 2.5ul of Exo I nuclease (NEB) was then added directly to each 'dirty' PCR product and incubated at 37°C for 1 hour, followed by purification using QIAquick PCR Purification Kit (Qiagen). Samples were further purified by 2% E-Gel SizeSelect agarose gel (Invitrogen). The resulting products were purified using QIAquick PCR Purification Kit (Qiagen), after which all samples were pooled at equimolar amounts (quantified by QuBit [Invitrogen]) and then quantitated by Bioanalyzer (Agilent).