Bar-seq of wild-type Saccharomyces cerevisiae: initial population, replicate 7
The library was created by PCR of UPTAGs using the primers UniBSeqF: 5Õ-AATGATACGGCGACCACC GAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNGATGTCCACGAGGTC TCT, and BSeqR: 5Õ-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCGACCTGCAGCGTACG, where N is a mixture of all bases and X is a unique 6 bp sequence used for multiplexing. PCR was carried out using ~50 ng/_l of template to produce a 186 bp PCR product using Ex Taq DNA polymerase (Takara). The thermocycling profile was 98 C for 10s followed by 30 cycles of 98 C for 10 s, 57 C for 30 s, and 72 C for 30 s, with a 10 min final extension. The products were purified using a Qiagen PCR purification kit before quantification using a Qubit fluorimeter and equal normalization.