TITLE: Extracellular DNA extraction
AUTHORS: Charline Giguet-Covex, Pierre Taberlet, Francesco Gentile Ficetola

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Over the past decade, an increasing number of studies has used environmental DNA from lake sediments to trace past lake ecosystem and landscape changes, agricultural activities or human presence and more broadly the biodiversity. In the environment, DNA can be found as intracellular and extracellular DNA (iDNA and exDNA). The contribution of each of these pools varies according to the environments, but exDNA often represents a high proportion of the total DNA (e.g. Vuillemin et al. 2017 and reference therein). Focusing the analyses on these different pools will lead to different community composition and structure of communities (e.g. Vuillemin et al. 2017). For plants, we propose to focus on the exDNA fraction to avoid the extraction of DNA from plant macro-remains, which might lead to an overrepresentation of these taxa and limit the detection of the other, “rarer” taxa. </div><div class = "text-block">The manipulation of ancient DNA is delicate, and the biases brought during the experiments can be multiple. Therefore, it is essential to work carefully, under strict laboratory conditions, with multiple controls and several replicates of samples or extraction or PCR (e.g. Fulton 2012).</div><div class = "text-block">This protocol details a sampling and extraction method of exDNA from sediments. This method was firstly developed for soils by Taberlet et al. 2012. It uses a phosphate buffer to desorb the DNA fragments from particles such as clays and then, the binding, wash and elution buffers from the NucleoSpin® Soil kit (Macherey-Nagel). Here we present a modified protocol from Taberlet et al. 2012. One modification consists in the addition of a concentration step (by using the amicon ultra centrigugal filter system) after the mixing of sediments with the saturated phosphate buffer. This step allow to increase the DNA yield. The quantity of phosphate buffer is usually based on the quantity of wet sediments (e.g. for 15g of wet sediment/soil, we add 15 ml of phosphate buffer). However, because lake sediments can have very different water content (depending on the composition and compaction) and because the exDNA is adsorbed onto particles, we now propose to base the phosphate buffer quantity on the dry weight of sediments. </div><div class = "text-block">    Fulton T., 2012. Setting Up an Ancient DNA Laboratory. Book Chapter, Ancient DNA in Methods in Molecular Biology edited by Shapiro B. and Hofreiter M., 1-11. </div><div class = "text-block">    Taberlet, P., Prud’Homme, S.M., Campione, E., Roy, J., Miquel, C., Shehzad, W., Gielly, L., Rioux, D., Choler, P., Clément, J.‐C., Melodelima, C., Pompanon, F. and Coissac, E, 2012. Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies. Molecular Ecology, 21: 1816-1820.</div><div class = "text-block">    Vuillemin A., Horn F., Alawi M., Henny C., Wagner D., Crowe S. A., Kallmeyer J., 2017. Preservation and Significance of Extracellular DNA in Ferruginous Sediments from Lake Towuti, Indonesia. Frontiers in Microbiology 8, 1440.</div></div>

[STEPS]
?. [exDNA extraction]
Phosphate buffer preparation:The phosphate buffer must be prepared the same day, before starting the extraction protocol. Calculate the quantity of phosphate buffer required for the extractions. Prepare a little more especially for the extraction control. You will add the same volume of phosphate buffer as 2 or 3 times the dry weight of sample depending on the quantity of sediment sampled and the water content. You will apply the same ratio for all samples of a same study. It is important to take care to not sample too much sediments to be able to add the phosphate buffer in the 50 ml falcon and avoid overflowing.Add the right quantities of NaH2PO4 and NA2HPO4 in the DNA-free water (To prepare   of phosphate buffer: add   of NaH2PO4 powder and  of NA2HPO4 powder) Mix until the total dissolution of the phosphate powder. Please be sure that the phosphate powder is completely dissoluted (i.e. no powder emains in the bottle)Add the right quantities of NaH2PO4 and NA2HPO4 in the DNA-free water (To prepare  of phosphate buffer: add  of NaH2PO4 powder and of NA2HPO4 powder) If you have, put in the UV box for
1 L
1970 mg
14700 mg
?. [exDNA extraction]
Addition of phosphate buffer to samples:Add the same volume of phosphate buffer to each 50 ml falcon. Each falcon should receive 2 or 3 times the dry weight of sediment (e.g. for  of dry sediment).Mix with an orbital shakerDuring the shaking time, label the final collection tubes, Amicons and green columns and prepare the tubes for SB ()
15 ml
5 g
200 µl
?. [exDNA extraction]
Centrifugations:Centrifuge the falcons to separate the sediment .Transfer  of the supernatant to the amicons and centrifuge for  intervals until concentrated down to about  in filter (the time for concentration depends on the sediment type). You can decide to centrifuge nearly all the supernatant (e.g.  ) or only a part. If the concentration is fast we suggest to centrifuge all the supernatant. On an aluminium foil (avoid notebooks in a clean lab) note the quantity of supernatant added and the quantity obtained after the centrifuge to know the concentration rate (it is generally difficult to obtain exactly the 400 µl required for the following steps. We usually oscillate between around 450 and 700 µl)Heat X ml (for X samples) of SE to  (in 1.5 or 2 ml tubes )
Centrifuge: 4400 33
50 ml
400 µl
12 ml
80 °C
?. [exDNA extraction]
DNA binding:Add the  of the concentrated extract in the tube previously filled with  of SB buffer, resuspend and transfer the  of supernatant to the green column (from NucleoSpin® Soil kit (Macherey-Nagel))centrifuge at . Discard flow-through. (You can also use the Quiavac system to save time)Prepare the control with  of phosphate buffer +  of SB buffer
400 µl
200 µl
600 µl
Centrifuge: 11000 33, 1 min
400 µl
200 µl
?. [exDNA extraction]
Wash silica membrane
?. [exDNA extraction]
1st wash:Add  SB on each column.Centrifuge at .. Discard flow-through.
500 µl
Centrifuge: 11000 33, 30 s
?. [exDNA extraction]
2nd wash: Add SW1 on each column.Centrifuge at  . Discard flow-through.
550 µl
Centrifuge: 11000 33, 30 s
?. [exDNA extraction]
3rd wash:Add correct volume of ethanol to SW2 - for 250 prep kit, add of ethanol to SW2 concentrate. (only FOR the FIRST USE of this BUFFER)Add  SW2 on each column.Centrifuge at . Discard flow-through.
400 ml
100 ml
700 µl
Centrifuge: 11000 33, 30 s
?. [exDNA extraction]
4th wash:Centrifuge at  .. Discard flow-through.
Centrifuge: 11000 33, 30 s
?. [exDNA extraction]
Dry Silica Membrane:Centrifuge
Centrifuge: 11000 33, 2 min
?. [exDNA extraction]
DNA elution:Place each column on a new  collection tube (with the sample label).Add  SE to each column. Incubate   (), lid open, followed by additional  with lid closed.Centrifuge at .Repeat with additional  SE for final elution volume of.Store at DNA is ready for quantification (we use the Qubit system or the Quantifluor® ONE dsDNA system from Promega) and PCR
1.5 ml
50 µl
0 Room temperature
Centrifuge: 11000 33, 30 s
50 µl
100 µl
-20 °C
?. [exDNA extraction]
Preparation of the SW2 buffer within the kit (NucleoSpin® Soil kit from Macherey-Nagel):This step is only FOR the FIRST USE of this BUFFERAdd correct volume of ethanol to SW2 (e.g. for 250 prep kit, add of ethanol to SW2 concentrate).
400 ml
100 ml