GSM2235554: Growth competition galactose; Saccharomyces cerevisiae; OTHER
Genomic DNA was extracted using YeaStar Genomic DNA Kit (Zymo Catalog # D2002). A fragment of 300bp, containing the strain and promoter barcodes, was amplified from the gDNA by PCR. In order to maintain the complexity of the library amplified from gDNA, PCR reactions were carried out on a total of 1200ng of gDNA, an amount calculated to contain >10^7 yeast genomes. To minimize erroneous representation of the strains in the final product due to PCR bias, 12 separate reactions of PCR were performed for each sample (each with 100ng gDNA) and reactions were limited to 24 cycles. PCR was performed with 3 alternatively barcoded 5' primers, used to barcode the different experimental timepoints. Primers also included 5 random nucleotides at their 5' to increase sequence complexity and facilitate cluster calling during sequencing. 51'- NNNNNCTACTTACGTGAACCATCACCCTAATCAA 52'- NNNNNCTACTTACGTGAACCATCACCCTAATCAA 53'- NNNNNTCCGATACGTGAACCATCACCCTAATCAA 3'- NNNNNCTTTGCCTTCGTTTATCTTGCC. Italic nucleotides denote the timepoint barcodes. Following PCR, products were united and separated from unspecific fragments by electrophoresis on a 2% agarose gel stained by EtBr, cut from the gel, and cleaned in 2 steps: QIAquick Gel Extraction Kit (Qiagen Catalog # 28704) and SPRI beads (Agencourt AMPure XP Catalog A63881). Concentration was measured using a monochromator (Tecan i-control). The samples were assessed for size and purity at the Tapestation, using high-sensitivity D1K screenTape (Agilent Technologies, Catalog # 5067-5582). 50 ng DNA were used for library preparation for next-generation sequencing; specific Illumina adaptors were added, and DNA was amplified using 8 amplification cycles (protocol adopted from Blecher-Gonen et al.). Samples were run on the NextSeq (Illumina) with 75bp single-end kits (Catalog # FC-404-1001). Illumina adaptors ligation.