PacBio seq of Salmonella ProQ mutatants: Pool 2 C
Mutations were introduced into the proQ ORF by error-prone PCR, and mutant proQ sequences were cloned in-frame with dTomato on an IPTG-inducible plasmid. The mutant library was then transformed into proQ Salmonella cells carrying the PfliE-gfp reporter and was physically sorted into three pools based on dTomato and GFP levels using fluorescence-activated cell sorting (FACS). Each pool of sorted cells was used as template in a PCR reaction with barcoded primers to generate sequences for high-throughput sequencing analysis. All samples were pooled, of which 500 ng was used for sequencing-library preparation. Sequencing libraries were constructed using the SMRTbellTM Template Prep Kit 1.0 (500 bp-20 kb) and purified with the AMPure Clean-Up kit (PacBio). The library quality was evaluated using a Bioanalyzer Chip (Agilent) before sequencing. Sequencing was performed on a SequelTM SMART Cell using the Sequell II system (PacBio). Library preparation and sequencing of the pooled DNA sample (barcoded amplicons) were performed at the Uppsala Genome Centre (UGC), a national facility within the National Genomic Infrastructure (NGI) hosted by Science for Life Laboratory (SciLifeLab) in Uppsala, Sweden.
SUB9259561
nonfunc-C