title: DNaseI digestion of T. kodakarensis chromatin
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). Cells were harvested from late log-stationary phase culture and cell suspensions (1010 cells per milliliter) were disrupted by the addition of extraction buffer (25 mM HEPES [pH 7.0], 15 mM MgCl2, 100 mM NaCl, 0.4 M Sorbitol, and 0.5% Triton X-100). After incubating for 10 min at 4°C, soluble proteins (supernatant) and a chromosomal DNA-enriched insoluble fraction (pellet) were separated by centrifugation at 14,000 g for 20 min. The insoluble chromatin fraction, containing DNA and chromatin-associated protein, was washed with the extraction buffer and frozen at -20°C until use. Chromatin pellet was digested with  0.1 units of DNaseI (Worthington Biochemical Corp., NJ, USA) for 1 hour at 37°C in the presence of 10 µg/µl RNase A 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. DNA fragments ranged from 10-100bp.     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.