solexa pair end sequence
The pooled deletion mutant cells were lysed in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) using a FastPrep-24 Instrument (MP Biomedicals). Genomic DNA was extracted from the lysate with the MasterPure Yeast DNA Purification Kit (EPICENTRE Biotechnologies). A primer extension reaction was carried out with Ex Taq HS DNA polymerase (TaKaRa) through 40 cycles of 30 s at 95°C, 15 s at 55°C, and 23 s at 63°C. For uptag sequencing sample preparation, 5’-GGTATTCTGGGCCTCCATGTCG-3’ was used as primer; for dntag sequencing sample preparation, 5’-TGTGAATGCTGGTCGCTATACTG-3’ was used as primer. The primer extension products were purified with GFX PCR DNA and Gel Band Purification kit (GE Healthcare) and ligated to a linker containing a 6-nt random 3’ overhang. The linker was prepared by annealing two synthetic oligonucleotides: 5’-GGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNN-NH2-3’, and 5’-P-AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG-NH2-3’. The ligation reaction products were purified with the GFX kit and used as templates for 30 cycles of PCR amplification. One PCR primer, 5’-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGA-3’, anneals to the linker sequence. The other PCR primer is a nested primer annealing to the sequences between the barcodes and the primer extension reaction oligonucleotides. For uptags, the nested primer was 5’-AATGATACGGCGACCACCGAGATCTACACTgggtgacccggcggggac-3’; for dntags, the nested primer was 5’-AATGATACGGCGACCACCGAGATCTACACTctgtcgattcgatactaacgcc-3’. These PCR primers contain sequences needed for Illumina paired-end sequencing in their 5’-end. The PCR products were run on an agarose gel and DNA in the size range of 250-300 bp was retrieved from the gel and purified with the GFX kit. The gel-fractionated DNA was amplified for 10 more cycles using the primers 5’-AATGATACGGCGACCACCGA-3’, and 5’-CAAGCAGAAGACGGCATACGA-3’. The amplified DNA was gel purified and used as sequencing template for paired-end sequencing with Genome Analyzer II (Illumina). For uptags, the read 1 sequencing primer was 5’-GGGTGACCCGGCGGGGACGAGGCAAGCT-3’; for dntags, the read 1 sequencing primer was 5’-CTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGA-3’. Standard Illumina sequencing primer was used for read 2.