Phenotype Sequencing of 24 pooled ppc- E. coli mutants
Bacterial genomic DNA was prepared from 24 mutant strains using the DNEasy kit from Qiagen using the optional RNAse treatments. The isolated genomic DNA from the mutant strains was pooled in 8 pools, each at a total concentration of 20ng/µL. Equal amounts of DNA from 3 mutant strains were mixed in each of the 8 pools. The pooled samples were then fragmented by sonication to an average size of 100–250 bp and confirmed by gel electrophoresis. 8 tagged genomic sequencing libraries (8 different indexes) were constructed using the TruSeq DNA Sample Prep Oligo Kit following the low throughput protocols provided by the manufacturer (Illumina). The final concentration of each of the 8 indexed libraries was measured by QuantiFluor assay and the 8 libraries were mixed in equal proportion at a final concentration of 10nM. 100bp single end read sequencing was carried out on a single lane of an Illumina Genome Analyzer HiSeq 2000 instrument by the UCLA Broad Stem Cell Research Center High Throughput Sequencing Facility.