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Table 8 Nucleic Acid Extraction Procedures As Described Elsewhere [7, 13]

From: Next-generation sequencing for hypothesis-free genomic detection of invasive tropical infections in poly-microbially contaminated, formalin-fixed, paraffin-embedded tissue samples – a proof-of-principle assessment

Common procedures for formalin-fixed, paraffin-embedded (FFPE) samples with histological diagnosis of invasive amebiasis and mycosis
 • Exposure of 25 μm thick sections in 1.5 ml tubes for 2 × 10 min to 1200 μl xylene and for 3 × 10 min to 1200 μl 100% ethanol under gentle constant agitation for deparaffination
 • Discarding of the supernatant following each 10-min step after centrifugation for 10 min at 13,000 g
 • Air-drying of the samples
Specific procedures as applied for the samples with invasive mycosis
 • DNA extraction using the DNA FFPE tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction as follows:
   Proteinase K digestion at 56 °C overnight to get a clear suspension
   Lysis of the fungal cell walls in the pellet with 400 units of Arthrobacter luteus lyticase L2524 (Sigma-Aldrich Corporation, St. Louis, MO, USA) (instead of lyticase L4276 [Sigma-Aldrich Corporation] which was no longer available) per sample for 45 min at 37 °C
Specific procedures as applied for the samples with invasive amebiasis
 • Tissue digestion in 200 μl lysis buffer (0.5% Tween 20 [ICI, American Limited, Merck, Hohenbrunn, Germany], 2 mg/ml proteinase K [Roche, Mannheim, Germany], 3.5 mM MgCl2, 15 mM ammonium sulfate, and 60 mM Tris/HCl [pH 8.5]) for at least 1 h until a clear lysate was obtained at 56 °C under continuous shaking
 • Boiling for 10 min to stop the digestion by denaturing the proteinase K
 • DNA extraction using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions