GSM5288833: Sort-seq; Escherichia coli K-12; OTHER
Minipreps were performed to isolate plasmid DNA from the cell pellets, and DNA concentrations were measured using a Qubit 4 Fluorometer (Invitrogen). Phusion PCR reactions (NEB) were used to generate amplicons from purified plasmid DNA. For each sample, two 50 μL reactions were performed using 100 ng of plasmid DNA template and 0.2 μM each of forward and reverse primers. Forward and reverse primers were used to introduce fluorescence bin-specific 10 bp barcode sites. These barcodes enabled multiplexed sequencing of samples from all sort-seq fluorescence bins.    After an initial denaturation step of 30 s at 98°C, 12 amplification cycles were performed of 10 s at 98°C, 30 s at 68°C, and 15 s at 72°C. A final elongation step was performed for 10 min at 72°C. For each fluorescence bin sample, the two PCR reactions were combined and purified using DNA purification spin columns (EconoSpin). DNA concentrations were measured using a Qubit 4 Fluorometer (Invitrogen) and samples for each fluorescence bin were combined in equimolar concentrations to generate one sample for NGS.    Subsequent sample preparation steps were performed at GENEWIZ, Inc. through their custom short-read amplicon service. DNA library preparation was performed using NEBNext Ultra DNA Library Prep kit following the manufacturer's recommendations (Illulmina). The DNA library was validated using TapeStation (Agilent Technologies) and quantified using Qubit 2.0 Fluorometer and qPCR.    DNA was loaded on the Illumina MiSeq instrument according to manufacturer's instructions and sequenced using a 2x250 paired-end configuration.