GSM5288832: Pre-sort; Escherichia coli K-12; OTHER
Plasmid DNA was purified from the sample using a Qiagen Plasmid Plus Midiprep kit and DNA concentration was 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 primer overhangs introduced partial Illumina adapters to enable compatibility with the GENEWIZ Amplicon-EZ service.    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. PCR reactions were combined and purified using DNA purification spin columns (EconoSpin).    Subsequent sample preparation steps were performed at GENEWIZ, Inc. through their Amplicon-EZ service. DNA library preparation, clustering, and sequencing were performed using NEBNext Ultra DNA Library Prep kit following the manufacturer's recommendations (Illumina). Adapter-ligated DNA was indexed and enriched by limited cycle PCR.    DNA was loaded on the Illumina MiSeq instrument according to manufacturer's instructions and sequenced using a 2x250 paired-end configuration.