title: 2b-RAD of Listeria monocytogenes: food isolates: Sample L4
design: "DNA was digested in 6-uL reaction volume using 1 U AlfI (Thermo Fisher Scientific) at 37 oC for 1 h. Enzyme heat inactivation was conducted at 65 oC for 20 min. The ligation reaction was performed by combining 5 uL of digested DNA with 12 uL of a ligation master mix containing 0.4 uM each of two library-specific adaptors (with fully degenerate cohesive ends (5'-NN-3'), 0.2 mM ATP (NEB), and 1000 U T4 DNA ligase (CABRU). Ligation was carried out at 16oC for 3 hour with a subsequent heat inactivation for 10 minutes at 65oC. Sample-specific barcodes were introduced by PCR with platform-specific barcode-bearing primers. Adaptor and primer sequences were those provided by Wang et al. 2012. PCR products were purified by using the SPRIselect purification kit (Beckman Coulter). Libraries were pooled and sequenced at the BMR Genomics facilities (Padova, Italy) by following a 96-plex 50SE strategy on a Miseq instrument (Illumina). Quality and adapters trimming of the sequenced reads was performed by running the customized script TruncateFastq.pl (http://people.oregonstate.edu/~meyere/tools.html)."