Yeast deletion pool Bar-Seq_OMIM
Genomic DNA was isolated from cells harvested at the end of pooled growth. Each 20-mer uptag barcode was amplified using composite primers comprised of the sequences of the indexing tag and the sequences of the common barcode primers: 5′-GNNNNNNGATGTCCACGAGGTCTCT-3′ (forward) and 5′-CNNNNNNGTCGACCTGCAGCGTACG-3′ (reverse). The 5′ portion (italics) is the variable sequence, which represents the 6-mer indexing tag used for multiplexing. The 3′ portion (bold) represents the common primer flanking the uptag barcode; it is required to amplify the yeast barcodes. PCR amplification was carried out at an annealing temperature of 55°C for 30 cycles using a DNA Engine Tetrad Peltier Thermal Cycler. The PCR products were gel-purified from 4% agarose gels. Equal volumes of normalized DNA were then pooled in one tube and sequenced using a Genome Analyzer (Illumina) according to the manufacturer's protocols