Supplementary Information
Fig. S1.
1
Fig. S2.
2
Fig.S3
3
Fig. S4.
4
Fig.S5.
5
Fig. S6.
6
Permafrost
AL
Depth (cm)
14C-age
BP
Cal BP
12±2
modern
modern
24±2
1230±35
1170±65
33.5±2.5
2450±30
2540±125
49±2
2790±30
2900±35
68±2
3190±40
3420±35
109±2
3650±35
3990±65
123±2
3710±35
4100±60
Table S1.
7
Precautions taken to prevent contamination of samples
During the extraction of nucleic acids and cDNA synthesis.
The PowerMax™ Soil Total RNA Isolation Kit (Mobio, Carlsbad, CA) was preferred for
permafrost DNA and RNA work for the stringent mechanical cell disruption step and to
minimize the chance of contamination by foreign nucleic acids and nucleases due to the use of
sterile buffers and disposable plastics.
Extraction of nucleic acids and enzymatic steps involved in cDNA synthesis were
performed inside a sterilized HEPA-filtered laminar flow bench in a PCR product-free, positivepressured, HEPA-filtered, and UV-sterilized clean lab dedicated for ancient DNA work.
Immediately prior to the extractions, the surfaces in the lab were cleaned with 6 wt% sodium
hypochlorite and Eliminase (Decon Laboratories, King of Prussia, PA) to remove any traces of
DNA and nucleases. Full body clean-lab suits were worn inside the clean lab and disposable
gloves were replaced frequently. Separate pipettes and sterile, DNA-free dual-filter tips
(Eppendorf) were used in order to prevent the introduction of foreign DNA via aerosols during
pipetting. As a control for contamination during DNA extraction, two reagent mixtures without
sediment were subjected to the entire extraction and purification procedure (extraction controls).
During preparations of PCR reactions. All PCR reactions were prepared in a HEPA-filtered
laminar flow bench dedicated for pipetting PCR reagents inside the clean lab. The PCR reaction
components and the various control reactions to test for contamination during the pipetting of the
reaction mixture components and during extraction of DNA were identical to those described
recently in (Coolen et al., 2009). The 96-well optical twin-tec plates (Eppendorf) with the PCR
reaction mixtures were sealed with optical film inside the clean lab. In order to prevent cross
contamination of samples and reactions with modern DNA, 1 µl of the standard series used to
calibrate bacterial, archaeal, and eukaryotic rDNA and cDNA copy numbers, was added to
reaction mixtures located in the first column of the plate and sealed off in a lab spatially
separated from the clean lab.
References
Coolen, M.J.L. and Shtereva, G. (2009) Metabolically active eukaryotes along a vertical nutrient
and redox gradient in the water column and sulfidic surface sediment of the Black Sea. FEMS
Microbiol Ecol 70: 525-539.
Coolen, M.J.L., Saenz, J.P., Giosan, L., Trowbridge, N.Y., Dimitrov, P., Dimitrov, D., and
Eglinton, T.I. (2009) DNA and lipid molecular stratigraphic records of haptophyte succession in
the Black Sea during the Holocene. Earth and Planetary Science Letters 284: 610-621.
Walker, D.A. and H.A. Maier (2008). Vegetation in the vicinity of the Toolik Field Station,
Alaska. Biological papers of the University of Alaska, No 28. Institute of Arctic Biology,
Fairbanks, AK.
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