title: GSM8316077: Combinatorial_1_input3; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Input and output pellets (3 biological replicates each, 6 tubes in total) were resuspended in 2ml extraction buffer (2% Triton-X, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA pH 8) and subjected to two cycles of freezing-thawing in an ethanol-dry ice bath and water bath at 62C for 10min each. 1.5ml of phenol:chloroform:isoamyl 25:24:1 was then added to the pellets and vortexed for 10min. The aqueous phase was recovered by means of 30min centrifugation at 3000 rpm. DNA was precipitated with 1:10 V NaOAc 3M and 2.2 V of 99% cold ethanol and incubated at -20C for 2h. After centrifugation, pellets were dried overnight at RT. The next day, pellets were resuspended in TE 1X buffer (10mM Tris pH8, 1mM EDTA pH8) and treated with 10ul RNase A (Thermo Scientific) for 1h at 37C. DNA was purified using a silica beads extraction kit (QIAEX II Gel Extraction Kit, Qiagen). PCR amplification in two steps using Q5 High-Fidelity DNA Polymerase (NEB). In step 1, 50 (for Combinatorial_2) or 100 (for N_terminal_Ab42_double_mutants, C_terminal_Ab42_double_mutants and Combinatorial_1 libraries) million plasmids were amplified for 15 cycles using frame-shifted adaptor primers with a partial homology to standard Illumina sequencing primers. PCR products from the first step were used as templates in the second PCR step, where indexed Illumina primers were used for a 10 cycles amplification. The three input samples were pooled together equimolarly, and the same for the three output samples. The two final pools were purified from a 2% agarose gel using a silica beads extraction kit (QIAEX II Gel Extraction Kit, Qiagen).