title: MNase digestion of T kodakarensis genomic DNA
design: T. kodakarensis strain KOD1 was cultivated under anaerobic conditions at 85°C in nutrient-rich medium ASW-YT, supplemented with 0.2% elemental sulfur (0.8x ASW-YT-S0) as described (Maruyama et al., 2011).Resulting DNA was treated with Proteinase K (1 mg/ml) for 2 hours at 37°C and purified by phenol-chloroform extraction and ethanol precipitation. The de-proteinised genomic DNA was then digested with 0.03 units of MNase (Worthington) to create a range of fragments from 10bp-1000bp.    Adapters (Illumina) were ligated to 4 µg of DNA fragments using the NEBNext DNA sample prep master mix set 1 and size selected on polyacrylamide gels to preserve the size distribution of the fragments (Kent et al., 2011). Ligated fragments were amplified (12 cycles) using Phusion DNA polymerase and adaptor-specific primers (Illumina), followed by purification using AMPure XP beads (Agencourt). 7 pM DNA was hybridized on one lane of an Illumina flowcell resulting in a clusters density of ~700 K/mm2 PF and sequenced using 100 nucleotide paired-end mode on an Illumina HiSeq 2000 using TruSeq SBS reagents version 3.