GSM9045626: libraryPDZ3_C_aPCA_G05_DC_a3_a1_output_bio_rep_3; Saccharomyces cerevisiae; OTHER
Cell pellets (one for each experiment input/output replicate) were re-suspended in 2 mL of DNA extraction buffer, frozen by dry ice-ethanol bath and incubated at 62ºC water bath twice. Subsequently, 2 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. 0.2 mL of NaOAc 3M and 4.4 mL of pre-chilled absolute ethanol were added to the aqueous phase. The samples were gently mixed and incubated at -20ºC at least for 30 minutes. After that, they were 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 (50 µL of QIAEX II beads, QIAGEN). The samples were washed twice with PE buffer and eluted twice by 125 µL of 10 mM Tris-HCI buffer, pH 8.5. 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. The sequencing libraries were constructed in two consecutive PCR reactions. The first PCR (PCR1) was designed to amplify the mutated protein of interest and to increase the nucleotide complexity of the first sequenced bases by introducing frame-shift bases between the adapters and the sequencing region of interest. The second PCR (PCR2) was necessary to add the remainder of the Illumina adapter and demultiplexing indexes. PCR2 reactions were run for each sample independently using Hot Start High-Fidelity DNA Polymerase. In this second PCR the remaining parts of the Illumina adapters were added to the library amplicon. The forward primer (5' P5 Illumina adapter) was the same for all samples in libraries 1-3 and containing a different barcode index in library 4. The reverse primer (3' P7 Illumina adapter) differed by the barcode index  to be subsequently pooled together and demultiplex them after deep sequencing. All samples were pooled in an equimolar ratio and gel purified using the QIAEX II Gel Extraction Kit. The purified amplicon library pools were subjected to Illumina 150bp paired-end Nextseq500 (libraries 1-3) or Nextseq2000 (library 4) sequencing at the CRG Genomics Core Facility.