BarSeq in Candida albicans: Collection of Overexpression mutants
To quantify the relative concentration of each barcoded strain in mixed populations, genomic DNA was prepared from 1 ml of OD600=50 culture, using the MasterPure Yeast DNA Purification Kit (Epicentre). Libraries were prepared following the 16S Metagenomic Sequencing Library Preparation protocol (Illumina, Inc., San Diego, United States) recommendations. In addition to the so-called Illumina overhang sequences, spacer bases (0, 4, 8, and 12 N) were added to the forward primers to shift the signal during sequencing to avoid mistakes during the sequencing due to the similar flanking regions of the BC amplicons. The purified amplicons, which contained the Illumina overhang, were then indexed with Illumina Nextera XT Index kit (Illumina, Inc., San Diego, CA, USA). Libraries were quantified with Qubit and then quality-checked and quantified on a Bioanalyzer (Agilent). The libraries were pooled in equimolar amounts and sequenced on an Illumina NextSeq500 Sequencing System with 75 bp single-reads.