Primers forward and reverse

Based on total DNA isolates, target regions were amplified using the primers listed in Table 2. The PCR of all plastid markers was carried out in accordance to the Shaw et al. (2007) (link) “slow and cold” protocol, whereas nuclear markers were amplified due to the Sabovljević & Frahm (2011) recommendations. In all cases the total volume of PCR mixture was 20 μL and comprised of REDTaq DNA Polymerase (0.05 U/μL) (Sigma-Aldrich, St. Louis, MO, USA), 1× REDTaq Reaction Buffer containing MgCl₂ (Sigma-Aldrich, St. Louis, MO, USA), primers forward and reverse (0.2 μm each primer) (Sigma-Aldrich, St. Louis, MO, USA), dNTPs solution (200 μm each dNTP) (Sigma-Aldrich, St. Louis, MO, USA), BSA (0.1 mg/mL) (New England BioLabs, Ipswich, MA, USA), one μL template DNA and water. The PCR products were run on agarose gel under the same conditions as DNA extracts (see above). All amplicon lengths included in the text are evaluated based on the mentioned gel electrophoresis (data not shown). I did not use any further improvement of selected PCR protocols, as long as my goal was to check the general feasibility of obtaining PCR-amplifiable DNA after Qiagen Kit and CTAB extractions.

Methylation status of the promoter region of NANOG was analyzed using Sanger sequencing after bisulfite conversion. Bisulfite conversion was performed using the innuCONVERT Bisulfite Basic Kit (Analytik Jena AG, Jena, Germany) according to the manufacturer's instructions using 500 ng of DNA.
PCR reaction contained 0.2 μl 5 μM primers (forward and reverse) (Sigma-Aldrich, Taufkirchen, Germany), 0.05 μl (5 units/μl) HotStarTaq Plus DNA Polymerase, 1 μl 10x PCR Buffer, 0.7 μl (3.33 mM) dNTPs, 6.35 μl ddH₂O (Qiagen, Hilden, Germany), and 15 ng bisulfite-converted DNA. Thermal conditions for PCR were as follows: 10 min at 95°C, 40 cycles of 1 min at 95°C, 1 min at 61°C, 1 min at 72°C, and final elongation for 10 min at 72°C. Primers used are listed in Additional file 1: Table S1.
Sanger sequencing was performed on SeqStudio Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). PCR products were directly used for a sequencing reaction after being purified with ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. The sequencing reaction was performed using Big-Dye terminator chemistry version 1.1 (Applied Biosystems, Foster City, CA, USA). Electropherograms were analyzed using the Sequence Scanner Software 2, Version 2.0 (Applied Biosystems, Foster City, CA, USA).

To determine TYMS mRNA levels, a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Barcelona, Spain) was used. The reaction was performed in a final volume of 20 μL, containing 1× SYBR Universal PCR Master mix (Applied Biosystems, Barcelona, Spain), 0.25 μM of reverse and forward primers (Sigma- Aldrich, Madrid, Spain), 3 μL of cDNA and H₂O mQ. PCR cycling conditions were 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The mRNA quantification was performed using the ΔΔCt method, where Ct is the threshold cycle that corresponds to the cycle where the amount of amplified mRNA reaches the threshold of fluorescence. Cyclophylin B (PPIB) was used as an endogenous control to normalize the results. Primer sequences for RT-qPCR are detailed in Table 1.