Apr 14, 2026
Version 2
USER II treatment of ancient DNA extracts V.2
- Alba Rey-Iglesia1,
- Deon de Jager1,
- Vanssy Li1,
- Andrea A. Cabrera1,
- Michael V. Westbury1,
- Eline D. Lorenzen1
- 1Globe Institute, University of Copenhagen
- Lorenzen Lab
Protocol Citation: Alba Rey-Iglesia, Deon de Jager, Vanssy Li, Andrea A. Cabrera, Michael V. Westbury, Eline D. Lorenzen 2026. USER II treatment of ancient DNA extracts. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6qrwgx9/v2Version created by Deon de Jager
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 13, 2026
Last Modified: April 14, 2026
Protocol Integer ID: 314932
Keywords: Ancient DNA, DNA extraction, USER treatment, UDG treatment, USER-II treatment, Uracil, Paleogenetics, antarctic thermolabile uracil dna glycosylase, thermolabile user ii enzyme, enzymatic removal of uracil base, lyase activity of endonuclease iii, enzymatic removal, user ii treatment of ancient dna, user enzyme, enzyme, uracil base, phosphodiester backbone, endonuclease iii, ancient dna fragment, ancient dna, dna
Abstract
Enzymatic removal of uracil bases from ancient DNA fragments. "Antarctic Thermolabile Uracil DNA glycosylase (UDG) catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease III breaks the phosphodiester backbone at the 3´and 5´ sides of the abasic site. In addition to generating a different a 3´-terminus than USER Enzyme (a 3´-phospho-α, β-unsaturated aldehyde versus the 3´phosphate left by USER Enzyme, NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) can also be completely heat inactivated after 10 minutes at 65°C." - https://www.neb.com/en/products/m5508-thermolabile-user-ii-enzyme?srsltid=AfmBOooXGOM1bvBPvVpj3P3CCKKOiRKUrQ-PfReg2m_cax4zrM3j8pN4.
Note
This protocol is the equivalent of a partial USER treatment, despite the 3 hr incubation which is usually associated with a full USER treatment. The reason this is a partial treatment is because it does not include PNK (polynucleotide kinase). PNK phosphorylates the 5' ends of DNA molecules/fragments. The UDG enzyme can only excise phosphorylated uracils. A small fraction of uracils at fragment ends exist in an unphosphorylated state. This means that they are not excised by UDG if PNK is absent, no matter how long the incubation is. This leads to the damage pattern observed for partial USER-treated libraries, which is what we observe in our libraries when using this protocol.
Note that the advantage of partial treatment is that one can use the same library for aDNA authentication via damage patterns and for analyses by clipping damaged bases at the ends of reads. This saves time and reagents in the lab. Full USER treatment should, at least theoretically, remove all aDNA damage, making distinguishing true endogenous ancient DNA and modern contaminant DNA tricky, if not impossible. If going the full USER treatment route, it is suggested to first generate a non-USER-treated library and checking via screening (low effort sequencing) that authentic endogenous ancient DNA is indeed present in the sample.
Protocol materials
Thermolabile USER II Enzyme - 250 unitsNew England BiolabsCatalog #M5508L
Monarch DNA Cleanup Columns (5ug) - 100 columnsNew England BiolabsCatalog #T1034L
Buffer PEQiagenCatalog #19065
1.5 mL LoBind tubes EppendorfCatalog #022431021
Buffer EBQiagenCatalog #19086
Before start
- UV-treat buffers (modified Buffer PB, Buffer PE, and Buffer EB/EBT) before starting for 10 min in a UV box.
- Do not UV Eppendorf/PCR tubes.
- PE buffer: Add Molecular Grade Ethanol as indicated on the bottle before use.
- Clean all surfaces with 5% bleach solution followed by 70% ethanol before and after use.
- Clean all equipment with 70% ethanol before and after use.
Buffer preparation
See following protocol for buffer preparation protocols.
Required buffers for this protocol:
- Buffer PE
- Modified Buffer PB
- Buffer EB - if using LoBind tubes
- Buffer EBT - if not using LoBind tubes (Buffer EB + Tween-20)
Incubation with USER II
In a PCR tube, add 4.8 µL Thermolabile USER II Enzyme - 250 unitsNew England BiolabsCatalog #M5508L to 27.2 µL DNA extract, for a final reaction volume of 32 µL.
Optional: Or add 2.4 µL USER II enzyme to 13.6 µL DNA extract, for a final reaction volume of 16 µL, if you want to keep some of the DNA extract as untreated.
In a thermocycler, incubate the reaction for 3 hours at 37°C then cool down to 10ºC. Can be left overnight at 10ºC.
Clean-up
Clean up using Monarch DNA Cleanup Columns (5ug) - 100 columnsNew England BiolabsCatalog #T1034L
Note
- Do not touch the film of the spin column when adding liquids.
- Pay attention when adding small volume of liquid. Make sure the liquid do not attach on the wall.
- As of 31 December 2024 the "Monarch DNA Cleanup Columns (5ug) New England Biolabs Catalog #T1024L" have been discontinued and replaced by "Monarch Spin Columns S1A and Tubes New England Biolabs Catalog #T2037L".
Add 450 µl modified Buffer PB to the spin column.
Add the entire USER II-DNA reaction (32 or 16 µL) and mix by pipetting or inversion.
Centrifuge at 8,000 RPM in a benchtop centrifuge for 1 min, discard flow through.
Add 650 µl Buffer PEQiagenCatalog #19065 to the spin column.
Centrifuge at 8,000 RPM for 1 min, discard flow through.
Centrifuge at max speed for 1 min to dry the column.
Place the spin column in a fresh 1.5 mL Eppendorf tube. Use 1.5 mL LoBind tubes EppendorfCatalog #022431021 , if available, but if not available, then use EBT buffer instead of EB buffer for the final elution (Step 4.8).
Add 20 µL Buffer EBQiagenCatalog #19086 to the spin column. If not using LoBind tubes, add 20 uL EBT buffer instead.
Incubate for 5 mins at room temperature to allow the DNA to elute from the column and dissolve in the buffer.
Centrifuge at max speed for 1 min.
Repeat Steps 4.8-4.10.
Discard the spin column and store the eluted DNA at -20ºC.
Protocol references
Hofreiter, M., Jaenicke, V., Serre, D., Haeseler, A. v., & Pääbo, S. (2001). DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Research, 29(23), 4793-4799. https://doi.org/10.1093/nar/29.23.4793