title: GSM8232392: C6_input_2 tech 3; Saccharomyces cerevisiae; OTHER
library_name: GSM8232392
library_construction_protocol: 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), and freeze-cracked by liquid nitrogen freezing followed by 62ºC incubation for 10 min in a 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. 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 vacuum-dried. 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 in 200 μL of 10 mM Tris-HCI buffer, pH 8.5. 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. The first PCR (PCR1) is used to amplify the amplicons for sequencing, to add a part of the Illumina sequencing adaptors to the amplicon and to increase nucleotide complexity for the sequencing reaction by introducing frame-shift bases between the adapters and the sequencing region of interest. The second PCR (PCR2) is used to add the remainder of the Illumina adaptor and to add demultiplexing indexes (dual-indexed). The amplicon libraries were run on a 2% agarose gel for quality control and quantification, and were pooled by block and size selected on a 2% agarose gel. Bands 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 at the CRG Genomics Core Facility.