TITLE: Minimally-invasive sampling of pars petrosa (os temporale) for ancient DNA extraction
AUTHORS: Eleftheria Orfanou, Marie Himmel, Franziska Aron, Wolfgang Haak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to obtain bone powder from the </span><span style = "font-style:italic;">pars petrosa</span><span> of disarticulated </span><span style = "font-style:italic;">ossis temporalis</span><span>, specifically from the dense parts around the cochlea, in a minimally-invasive way by drilling from the outside. </span></div><div class = "text-block"><span>The </span><span style = "font-style:italic;">pars petrosa</span><span> has been shown to consistently yield high amounts of aDNA (Pinhasi </span><span style = "font-style:italic;">et al.</span><span> 2015 </span><span style = "font-style:italic;">PLoS One, </span><span>doi: </span><a href="http://dx.doi.org/10.1371/journal.pone.0129102" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1371/journal.pone.0129102</span></a><span>).</span></div></div>

[STEPS]
?. [Workstation preparation]
Place a sheet of aluminium foil under the hood.Place weighing paper, drill bits, and forceps on a sheet of aluminum foil on an easily accessible clean surface outside of the hood. Alternatively, you may place them within hood but be sure to cover them with aluminum foil or a paper towel to protect them from dust produced during sampling.You will also need:2 ml Safe-Lock Biopur tubesWeighing trays/pans (optional)Clean and labelled sample bags according to the amount of samplesSmall pieces of aluminium foil for wrapping the boneSmall tape strips to fix the aluminium foil while covering the bonePaper towelsDiluted commercial bleach solution (1:10)UV-irradiated filtered/deionised water
Everything that comes in contact with the sample needs to be decontaminated in order to avoid cross-contamination between samples. Change gloves regularly, especially between different samples.
?. [Preparation of sample]
Place a sheet of aluminium foil in the UV chamber.Place the pars petrosa on the aluminium foil and place the labelled sample bag next to it (to avoid mixing up samples in case you work on more than one). UV- irradiate the bone for  on each side.
If possible, avoid touching the bone directly. Use precision forceps or something similar to handle each bone individually.
Tip: It is possible to UV irradiate multiple bones at once in the same UV chamber. Ensure each bone is on a separate bit of foil, and keep the corresponding labelled sample bag next to it to prevent sample mix up.
?. [Preparation of sample]
Take a new 2 ml tube and label it accordingly. If this is the beginning of the sampling session, tare the precision scale with the tube placed in the center.Label a new sample bag with the sample ID.
To avoid contamination, tubes should remain closed except when the bone powder is added.
?. [Preparation of sample]
Remove the bone from the UV chamber by using a paper towel with a small amount of household bleach solution.Wipe the surface of the bone region you want to sample with a bleach-dampened paper towel and then carefully clean the bone with a water-dampened paper towel to remove excess bleach. Use another sheet to wipe the bone dry.
If the bone was not washed after excavation this can be a tedious step. Make sure you brush/shake off as much of the residual soil as possible before starting.
?. [Preparation of sample]
Take a weighing tray with a new pair of gloves.Prepare a sheet of weighing paper by creasing it mid-way and place it under the area where you want to sample.
Bone powder can be statically charged and difficult to transfer to tubes. By slightly folding the weighing paper, it makes it easier to move the bone powder from the weighing paper into the tube.
?. [Sampling procedure]
Minimally-invasive sampling of pars petrosa
?. [Sampling procedure]
Familiarize yourself with the anatomy of the petrous bone (e.g., Pinhasi et al. 2015). Locate the dense parts around the cochlea (b & c) as shown below in Figure 2. Ideally you will target area c (orange) shown in Figure 2, which is located roughly halfway between the jugular fossa (bottom) and the petrous crest (top). However, since this protocol does not cut the petrous bone in half, it will be easier to orient the bone so that your are looking at the posterior part, which contains the internal acoustic opening (meatus acusticus internus). To target the cochlear region you will have to position the drill a few millimetres laterally (the outer side where the temporale is/would be attached) and choose an angle so that the drill hole will not penetrate the auditory canal (Figure 3). Of note, the auditory canal also leads laterally/dorsally outwards, so it is advisable to drill in a parallel line.
?. [Sampling procedure]
To prevent residual dust/dirt from the outer surface and crevices to contaminate your freshly drilled bone powder, you can wrap the sample with aluminium foil and/or tape. Leave the part that you want to drill unwrapped. For ease of orientation, mark the position of the internal auditory meatus with a permanent marker (Figure 4).
?. [Sampling procedure]
Use the drill to clean (abrade) the surface of sample window at low speed.Discard or set aside the powder from this cleaning step and place a new weighing tray and/or weighing paper under the sample.
?. [Sampling procedure]
Drill at low speed (and high torque) from the outside towards the cochlear region in parallel to the auditory canal.Collect clean bone powder on the weighing paper. Of note, it is very likely that you will drill into internal canals and porosities that can be filled with soil. If so, stop and try to reorient your drilling angle.Empty the drilling hole by carefully tipping it out onto the collection paper.
Pro-Tip: If residual dirt/soil is mixed in with your collected clean bone powder, you can physically separate them by carefully tipping the edges of the weighing paper repeatedly. In doing so, the different weights and particle sizes will easily separate. Discard or set aside the dirt.
Be careful when unwrapping the bone,  the vibration of the drilling will loosen residual dirt/soil and bone powder, which will accumulate in the wrapping.
?. [Weighing of bone powder]
Transfer the clean powder bone into a labelled 2 ml Safe-Lock Biopur tube. The folded weighing paper will help guide the powder into the tube.Close the tube, wipe it with a damp paper towel and weigh the tube.Write down the weight in mg on the cap and on the side of the tube.Store the bone powder at until further processing.
Bone powder can become statically charged. In such case, consider using an anti-static gun and apply to the weighing paper and tube.
Ideally you wish to collect around  to   of powder for extraction (if using our protocol for ancient DNA Extraction). If you have more than   then store it as back-up material in a separate tube.
-20 °C
?. [Weighing of bone powder]
Take photos after sampling for documentation. Put bone back into a new, UV irradiated, labelled bag.
?. [Decontamination]
Carefully clean the workspace and the equipment before sampling the next sample:
Everything that came in contact with the sample needs to be cleaned/decontaminated in order to avoid cross-contamination between samples.
?. [Decontamination]
Throw away disposable material such as aluminium foil and weighing tray/pan.Clean surfaces and tools such as pliers with bleach solutionUse less aggressive DNA decontamination reagents (e.g. DNA away) for sensitive material such as electronic devices (e.g. handpiece of the drill, precision balance). Wipe off bleach with water-wet paper towels afterwards to prevent corrosion; air dry.
?. [Decontamination]
Drill bits can be reused after careful cleaning: incubate them in bleach solution (1:10 dilution) for at least 2 minutes.       You can use a UV irradiated and bleached toothbrush for brushing the saw blade. Clean with UV irradiated water to remove all bleach and let it dry.      You can use a UV irradiated and bleached toothbrush for brushing the saw blade.
Check conditions of drill bits after ~5 uses and replace with new if necessary.
?. [Decontamination]
(or in case of UV irradiation of multiple samples at once,  )and repeat the sampling procedure with the next sample.