tn-seq of Akkermansia muciniphila
The bacterial genomic DNA was isolated by a genomic DNA extraction kit (Sigma Aldrich). One microgram of mutant library genomic DNA was digested for 1 hr at 37C with MmeI. The digestion products were dephosphorylated with calf intestine alkaline phosphatase (Invitrogen) for 30 min at 50C. The samples were barcoded using 6 bp barcodes, and DNA suitable for sequencing was generated by PCR as described previously. In this experiment, NEBNext High-Fidelity 2 PCR master mix (New England Biolabs) was used for PCR amplification