GSM8229602: pdz3cript_b1_input_biolrep3; Saccharomyces cerevisiae; OTHER
Cell pellets (one for each experiment input/output replicate) were re-suspended in 1 mL of DNA extraction buffer (2% Triton-X, 1% SDS, 100mM NaCl, 10mM Tris-HCl pH8, 1mM EDTA pH8), frozen by dry ice-ethanol bath and incubated at 62ºC water bath twice. Subsequently, 1 mL of Phenol/Chloro/Isoamyl 25:24:1 (equilibrated in 10mM Tris-HCl, 1mM EDTA, pH8) was added, together with 1 g of acid-washed glass beads (Sigma Aldrich) and the samples were vortexed for 10 minutes. Samples were centrifuged at RT for 30 minutes at 4,000 rpm and the aqueous phase was transferred into new tubes. The same step was repeated twice. 0.1 mL of NaOAc 3M and 2.2 mL of pre-chilled absolute ethanol were added to the aqueous phase. The samples were gently mixed and incubated at -20ºC for 30 minutes. They were then centrifuged for 30 min at full speed at 4ºC to precipitate the DNA. The ethanol was removed and the DNA pellet was allowed to dry overnight at RT. DNA pellets were resuspended in 0.6 mL TE 1X and treated with 5 uL of RNaseA (10mg/mL, Thermo Scientific) for 30 minutes at 37ºC. To desalt and concentrate the DNA solutions, QIAEX II Gel Extraction Kit was used (30 μL of QIAEX II beads). The samples were washed twice with PE buffer and eluted twice by 125 μL of 10 mM Tris-HCI buffer, pH 8.5 and then combined two elution. Finally, plasmid concentrations in the total DNA extract (that also contained yeast genomic DNA) were quantified by qPCR using the primer pair oGJJ152-oGJJ153, that binds to the ori region of the plasmids. We performed 2 consecutive PCR reactions for each sample (PCR1 and PCR2). In PCR1, we added frameshifting oligonucleotides and amplified the regions of interest (amplicon with constant region and mutated region) from the extracted DNA with 5 cycles. In PCR2 we added Illumina sequencing barcodes with PCR using the minimum number of cycles necessary to reach amplification plateau for each sample based on a qPCR run. The amplicon library pools were run on a 2% agarose gel and were purified using QIAEX II Gel Extraction Kit (QIAGEN) and using 30uL of QIAEX II beads for each sample. The purified amplicons were subjected to Illumina 150bp paired-end NextSeq sequencing. PCR amplicon