title: GSM8244567: fyn_permissivity_output2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Input and output culture cell pellets were resuspended in DNA extraction buffer (10 mM Tris-HCl, 100 mM NaCl, 2% Triton-X, 1% SDS, pH 8). Volumes were scaled depending on the volume of cell pellets to process. For pellets from 100 mL cultures harvested at OD600 1.6, 1 mL of DNA extraction buffer was used. Cells were lysed by cyclically freezing in liquid nitrogen followed by incubation in a 62ºC water bath twice. Total DNA was extracted by adding 1 mL of a 25:24:1 phenol:chloroform:isoamyl alcohol mixture (Merck, Darmstadt, Germany) along with 1 g of acid washed glass beads (Sigma-Aldrich, Saint Louis, MO, US) and vortexing for 10 min. The mixture was centrifuged at room temperature for 30 min at 3,300 g and the aqueous phase (upper layer) was recovered in a separate tube. 1 mL of the phenol:chloroform:isoamyl alcohol was added to it and the mixture was vortexed for 2 min followed by 45 min centrifugation at 3,300 g. The aqueous phase was again transferred to a new tube and mixed with 0.1 volumes of 3 M sodium acetate and 2.2 volumes of pure ethanol previously cooled down at -80ºC. The mixture was kept at -20 ºC for 30 min and spun down at 3,300 g for 30 min at 4ºC. The DNA pellets were dried overnight in a fume extraction hood.  Plasmid DNA from total DNA extracts was quantified by qPCR using the oGJJ152-oGJJ153 oligo pair that anneals at the origin of replication of all pGJJ vector series. The qPCR reaction was performed in a LightCycler 400 instrument (Roche, Basel, Switzerland) using the SYBR green qPCR 2X master mix from Thermo Fisher (Waltham, MA, US). Quantification was achieved by measuring a standard curve on a serial dilution of a control sample of known concentration in the same qPCR run as the query samples, and subtracting blank measurements.  Library preparation for sequencing in Illumina instruments followed a two-step PCR protocol. Frameshifts and a segment of the Illumina adapter overhangs were introduced in PCR1. For each sample, 8x50 μL PCR1 reactions were performed using 31.25 million plasmid molecules as a template as quantified by qPCR. PCR1 used the oGJJ595 and the oGJJ748 frameshifting oligo pools and consisted of 11 cycles. Next, all reactions corresponding to the same sample were pooled and treated with 16 μL ExoSAP for 30 min at 37ºC, then for 20 min at 80ºC. Samples were cleaned-up using MinElute columns, eluted in 30 μL of pre-warmed water (55ºC) and 1.5 μL were used as template for each PCR2 reaction.16x50 μ