DNeasy UltraClean 96 Microbial Kit (384)

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Publication protocol

To isolate in vivo-adapted EcN isolates, intestinal contents or fecal samples were vortexed in phosphate-buffered saline and streaked on LB plates containing kanamycin. Single colonies were placed in liquid media and grown at 37 C overnight. Un-adapted EcN strains were also grown overnight. Total DNA was extracted from each culture using the DNeasy UltraClean 96 Microbial kit (10196-4, Qiagen), and prepared for whole-genome sequencing using Nextera Tagmentation (Baym et al., 2015). Briefly, gDNA was brought to a concentration of 0.5 ng/mL in 1 mL volume. To each sample, 1.25 mL TD buffer, 0.125 mL TDE1 enzyme, and 0.125 mL nuclease-free water was added, and incubated at 55 C for 15 m. Then, 11.2 mL KAPA HiFi master mix was added to each tagmented sample. Indexed sequencing adaptors (5 mM each) were thawed, and 8.8 mL was added to each sample. Then, PCR was performed using the following protocol: 72 C for 3 m, 98 C for 5 m, 98 C for 10 s, 63 C for 30 s, 72 C for 30s, return to step 2 13 times, 72 C for 5 m, 4 C forever. PCR reactions were purified using AMPure XP beads according to the manufacturer’s protocol, and eluted in 60 mL resuspension buffer. Sequencing libraries were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Fisher Scientific), and pooled at equimolar concentrations for sequencing. 2 million 2x150bp sequencing reads were obtained per sample.

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