Dnase 1 buffer 10

The volume of each RNA extract was adjusted to 2 μg for each template. Firstly, RNA extracts were treated with DNase I (1 U/μL, Fermentas, Hanover, MA, USA) in presence of DNase I buffer 10× (Thermo Scientific) with MgCl₂ for 30 min at 37 °C. Subsequently, EDTA (Fermentas) was added for 10 min at 65 °C. The reverse transcription reaction contained 5× reaction Buffer (Fermentas), Riboblock Inhibitor (20 U/μL, Sigma-Aldrich), Universal RNA Spike II (0.005 ng/μL, TATAA biocenter, Göteborg, Sweden), 10 mM dNTP Mix (Thermo Scientific), oligo(dt)18 (Thermo Scientific) mixed 1:1 with Random primers (Thermo Scientific) and M-MuLV RevertAid transcriptase (200 U/μL, Fermentas), and run for 60 min at 42 °C followed by 10 min at 70 °C to generate cDNA. Obtained cDNA was stored at −20 °C. All cDNA samples were synthesized in duplicates. Negative control RT⁻ was prepared in the same conditions but with RNase/DNase free water (Thermo Scientific) instead of the Reverse transcriptase.

Total RNA was isolated from testicular fractions prepared by elutriation and testes samples using TRI Reagent (Sigma-Aldrich). Firstly, RNA extracts (2 μg) were treated with DNase I (1 U/μL, Fermentas, Hanover, MA) in presence of DNase I buffer 10× (Thermo Scientific) with MgCl₂ for 30 min at 37 °C and EDTA (Fermentas) was added for 10 min at 65 °C. The reverse transcription reaction contained 5x reaction Buffer (Fermantas), Riboblock Inhibitor (20 U/μL, Sigma-Aldrich), Universal RNA Spike II (0.005 ng/μl, TATAA biocenter, Sweden), 10 mM dNTP Mix (Thermo Scientific), oligo(dt)18 (Thermo Scientific) mixed 1:1 with Random primers (Thermo Scientific) and M-MuLV RevertAid transcriptase (200 U/μL, Fermentas), and run for 60 min at 42 °C followed by 10 min at 70 °C to generate cDNA.
For q-RT-PCR cDNA (10 ng/µl), two times Maxima SYBR Green qPCR Master Mix (Thermo Scientific), reverse and forward primer (1 μM, Generi Biotech, Hradec Kralove, Czech Republic) and nuclease free water were used.
The Ribosomal protein S2 (Rps2) gene was used as the reference gene. Specific gene markers for germinal cells and somatic cells were selected to determine elutriation fractions (the enrichment of individual elutriation fractions is listed in the Supplementary Table 1).