Barcode-seq of yeast double transporter deletion library treated with N-Phenylanthranilic Acid
The transporter double-deletion library pool was grown in yNB+Sc for 12 hours at 30C with agitation. Pools were then diluted to OD600 0.1 in 500 L of yNB+Sc containing the xenobiotic compounds in 48-well flat bottom plates. After 24 hours growth at 30C with agitation (approximately 5 generations), OD595 was measured and cultures were diluted 20x in 500 L of fresh media and allowed to grow for 12 hours (ca. 10 generations) under the same conditions. The dilution procedure was repeated and cultures were allowed to grow until stationary phase (ca. 15 generations). Cell pellets were collected and genomic DNA was extracted using Wizard Genomic DNA Purification Kit. Upstream barcodes were PCR amplified with U1 and U2 primers containing Illumina pre-adaptors for multiplex barcode sequencing with Illumina HiSeq2500 platform by the University of So Paulo Functional Genomics Center.