Biolink dna crosslinker

DNA isolation and library preparation were carried out at the Molecular Anthropology and Paleogenetic Laboratory of the Department of Biology, University of Florence, using facilities exclusively dedicated to ancient DNA analysis, following stringent protocols to prevent contamination with present-day DNA⁴¹ . Negative controls were included in each experimental step. The right intermediate cuneiform bone and a small portion of mummified liver tissue were collected for DNA analysis. To remove potential contaminants, the outer layer of the bone sample was brushed with disposable tools and irradiated with ultraviolet light (254 nm) for 45 min in a Biolink DNA Crosslinker (Biometra). Bone powder was then collected from the densest part of the bone using a low-speed dental micromotor equipped with disposable tungsten carbide ball burrs. DNA was extracted from 50 mg of bone powder using a silica-based protocol that allows DNA molecules to be recovered efficiently even if highly fragmented^{9 (link)}. In the final step, DNA was eluted twice in 50 µl TET buffer (10 nM Tris, 1 mM EDTA, 0.05% Tween-20). DNA was extracted from 50 mg of mummified liver tissue using the QIAamp DNA mini kit according to the manufacturer’s recommendations (Qiagen).

Only the small 3rd metatarsal 3150 from layer 4c of Grotta Paglicci (direct ¹⁴C date: 14,372–13,759 cal. yr bp) was selected for aDNA analysis. The oldest specimens are too small to extract enough quantity of bone powder without significantly damaging them. DNA analysis was carried out in the Molecular Anthropology Laboratory of the University of Florence, exclusively dedicated to ancient DNA analysis. Blanks as negative controls were used in all of the experimental steps to monitor the absence of contaminants in reagents and environment. To remove potential contamination, the outer layer of the bone was mechanically taken out using a dentist drill with disposable tip. After brushing, sample was irradiated by ultraviolet light for 45 min in a Biolink DNA Crosslinker (Biometra). The DNA was extracted from approximately 50 mg of bone powder following a published silica-based protocol^{50 (link),80 (link)} and eluted in 100 µl of TET buffer (10 nM Tris, 1 mM EDTA and 0.05% Tween-20). 20 μl of DNA extract were transformed into genetic library following a double-stranded DNA protocol^{81 (link)} using a unique combination of two indexes. Sample and negative controls were checked with Agilent 2100 Bioanalyzer DNA 1000 chip. Libraries were then enriched for mitochondrial DNA following a capture protocol^{81 (link),82 (link)} and sequenced on an Illumina MiSeq run for 2 × 75 + 8 + 8 cycles.