Qiavac 24 plus

We performed extractions in 6 sets, adding in an extraction blank (extraction reagents added to an empty 5 mL tube with no filter, n = 6) for each extraction set. Samples were randomized prior to extraction. We extracted DNA from each filter using the DNeasy Blood and Tissue Kit (Qiagen, USA) following the manufacturer’s protocol with the following modifications. We added 850 μL of lysis buffer [37 ], 100 μL of SDS (final concentration (C_f) = 1%), and 100 μL of proteinase K (Qiagen, USA) (C_f = 1 mg/mL) to each filter and incubated at 56°C for 14–16 hours. After incubation, we added 1 mL of Buffer AL (Qiagen, USA) and incubated at 56°C for 10 minutes. Then we added 1 mL of 100% molecular grade ethanol and mixed thoroughly by vortexing. We loaded the lysate from each filter into spin columns and used a QIAvac 24 Plus (Qiagen, USA) vacuum manifold. Luer plugs were soaked in 10% bleach and rinsed with deionized water before each use. After loading the 3 mL of lysate, we followed the DNeasy Blood and Tissue Kit protocol. We performed 2 elutions of 50 μL each for a total extract volume of 100 μL. We immediately quantified total DNA using the QUBIT DSDNA HS ASSAY (Invitrogen, USA) and stored extracts at -20°C until amplification within 1 month of extraction.

ctDNA was extracted and purified using Qiagen’s QIAamp^® circulating nucleic acid kit and QIAvac24 plus (Qiagen, Hilden, Germany) with modifications to the manufacturer’s protocol, as indicated by Sysmex Inostics.

cfDNA was extracted from aliquots (500 µl) of serum using the QIAamp circulating nucleic acid kit (Qiagen, Hilden, Germany) with the QIAvac 24 Plus vacuum manifold, following the manufacturer’s instructions. cfDNA purity was checked using an Agilent High Sensitivity DNA Kit and the Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA). When required, additional purification was performed using Agencourt AMPure XP (BeckMan Coulter, Brea, CA) to remove larger contaminating nucleic acid. cfDNA concentration was quantified with a Qubit 2.0 Fluorometer using the Agilent High Sensitivity DNA Kit (Agilent Technologies).
Tissue genomic DNA (gDNA) was extracted from formalin-fixed, paraffin-embedded (FFPE) tissues with the QIAamp DNA FFPE Tissue kit (Qiagen) according to the manufacturer’s instructions and eluted in a 50 μL volume. Purity of the extracted genomic DNA was assessed by electrophoresis of the DNA through a 1% agarose gel, and DNA concentration was quantified with a Qubit 2.0 Fluorometer using the Agilent High Sensitivity DNA Kit (Agilent Technologies).

Genomic DNA (gDNA) and total RNA were isolated from cell lines using TRIzol (Invitrogen) according to the manufacturer's protocol. gDNA was also isolated from formalin-fixed paraffin-embedded (FFPE) colorectal tissues with the AllPrep DNA/RNA FFPE Kit (Qiagen). cfDNA was isolated from 2–4 mL of plasma using the QIAamp® Circulating Nucleic Acid Kit (Qiagen) and the vacuum system QIAvac 24 Plus (Qiagen) following the manufacturer's recommendations. The quality and quantity of gDNA and RNA were evaluated with a NanoDrop (Thermo Fisher), and cfDNA was quantified by the QuantiFluor® ONE dsDNA kit with the Quantus™ Fluorometer (Promega). DNA and RNA were stored at − 80 °C until analysis.

Fragments about 1 cm² in size are sawed out of the middle of the diaphysis of the left femora. All outer surfaces of the fragments are removed to minimize the risk of a contamination with modern human DNA from, e.g., the excavation personal. The fragments are crushed with a steel mortar before they are powdered in a swing mill for 3 min at 24 swings per second. Afterwards, 0.25 g of the powder is transferred into a 15 ml FalconTube and 3900 μl of EDTA UltraPure™ 0.5 M pH 8 (Invitrogen™) and 100 μl of Proteinase K (600mAnson-U/ml) are added. This mixture is incubated for 18 h at 37 °C in a rotator. Now, an additional 50 μl of Proteinase K are added and the mixture is incubated at 56 °C for 2 h in a rotator. 50 μl of SDS (10 mg/ml) are added, followed by an incubation time of 5 min at 65 °C. The lysate is centrifuged at 3300rcf for 3 min to sediment surplus organic material. The lysate is transferred into a 50 ml FalconTube that contains 16 ml PB-Buffer (Qiagen) and 100 μl sodium acetate buffer (pH 5.2, 3 M, Sigma). After manually mixing the lysate, it is centrifuged at 3300rcf for 3 min. The DNA clean-up is conducted with minElute spin columns and funnels for large volumes using the QIAvac-24-plus (Qiagen). Deviating from the protocol, three wash steps with PE-buffer (Qiagen) are performed. The DNA is eluted in 60 μl RNase-free water (also cf. [54 ]).