Heritage Cases

Extraction: For DNA extraction, we will perform several incubations on the samples. At each incubation the samples will be kept in rotation. First, the samples will be pre-digested at 45 °C for 15 min in 1000 μL of extraction buffer, consisting of Urea, EDTA (0.5 M) (VWR) and 10 μL of Proteinase K (10 mg/mL) (VWR). A negative control will be added during this step and taken through the work process. The supernatant from the predigestion step will be removed and a fresh extraction buffer (same as above) with proteinase is added to the samples and left for digestion overnight at 37 °C. The supernatant from this step will be stored, and more extraction buffer and proteinase will be added to the samples (same as above) and left rotating at 55 °C for 4 h. The final supernatant will be collected and combined with the previous one (around 2000 μL) and spun down to 100 μL using membrane filters (Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-30 from Millipore). The extract will be purified using MinElute spin columns and a buffer set (both Qiagen). We will modify the Qiagen protocol (reducing the PE buffer volume to 600 μL and performing two elutions using 55 μL of the EB buffer) and obtain about 110 μL of extract, which will be stored at −20 °C. After extraction the DNA is captured on a silica membrane of a spin column and purified, resulting in 100 μL of product which is then stored at −20 °C. Libraries and sequencing: Double-stranded blunt-end libraries will be built using the modified protocol by Meyer and Kircher (2010). 20 μL of the extract will be used to build the blunt-end repair libraries and 30 μL for the USER enzyme pre-treated libraries. The master mix for the blunt end repair step will contain 4 μL of Buffer Tango 10× with BSA (Thermo Scientific), 0.16 μL 25mM dNTP mix (Thermo Scientific), 0.4 μL ATP 100mM 25 μmol (Thermo Scientific), 12.64 μL ddH2O, 2 μl T4 Polynucleotide Kinase (10 U/μL) (Thermo Scientific) and 0.8 T4 DNA Polymerase (5 U/μL) (Thermo Scientific). It will be incubated for 15 min at 25 °C, and then 5 min at 12 °C. MinElute spin columns will be used to purify the product, reducing the proposed volume of the washing buffer PE to 600 μL. The ligation master mix contains 10 μL ddH2O, 4 μL 10× T4 DNA ligase buffer (Thermo Scientific), 4 μL PEG4000 50% (w/v) (Thermo Scientific), 1 μL of Adaptor mix P5/P7 100mM (10 pmol) (Biomers.net) and 1 μL of T4 DNA ligase 5 weiss U/μL (Thermo Scientific), and further incubation will take place at 22 °C for 30 min. The adaptors will then be filled with the following master mix: 14.1 μL of ddH2O, 4 μL of 10× ThermoPol reaction Buffer (BioLabs), 0.4 μL of 25mM dNTP mix (Thermo Scientific) and 1.5 μL Bst DNA Polymerase Large Fragments 8000 U/mL (BioLabs). Incubation takes place at 37 °C for 20 min, followed by 20 min at 80 °C. Then UDG treatment is carried out before blunt-end repair for several libraries. We use USER Enzyme 1000 U/mL (BioLabs) and incubate the extract with the ingredients for a blunt-end mastermix (excluding polymerase, which is added after the incubation) for 3 h at 37 °C. We then proceed with the blunt-end protocol. Libraries are then amplified with 10 μM index primers (Biomers.net), using AmpliTaq Gold 1000 Units 5 U/μL (Applied Biosystems) for blunt-end libraries, and AccuPrime™ Pfx DNA Polymerase (2.5 U/μL) (Invitrogen) polymerase for damage repair libraries. We determine the number of cycles using quantitative PCR, with reagents from Thermo Scientific and Biomers. Libraries are sequenced on the Illumina Hiseq X platform at the SciLife centre in Stockholm, Sweden. References: Kashuba, N., Kırdök, E., Damlien, H. et al. Ancient DNA from mastics solidifies connection between material culture and genetics of mesolithic hunter–gatherers in Scandinavia. Commun Biol 2, 185 (2019). https://doi.org/10.1038/s42003-019-0399-1 Meyer, M. & Kircher, M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb. Protoc. (2010). https:// doi.org/10.1101/pdb.prot5448 PMID:20516186.