Encyclo polymerase mix

Total RNA extraction and purification from TERA1 and NT2/D1 cell lines, testicular cancer and normal testis samples were performed as described above. First strand cDNA synthesis was carried out with the random hexanucleotide primer (Promega, USA) and MintReverse Transcriptase (Evrogen, Russia) according to the manufacturers' protocols. For RT-qPCR, reactions were performed using qPCRmix-HS SYBR system (Evrogen, Russia) on Lightcycler 480 (Roche, USA) in accordance with the manufacturers' instructions. DNA fragments were amplified for 40 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 20 s. Relative level of mRNA was quantified with 18S rRNA serving as the reference. Technical triplicates were used to ensure reproducibility. For RT-PCR, reactions were performed using Encyclo Polymerase Mix (Evrogen, Russia) on Biorad DNAEngine PTC (Biorad, USA). DNA fragments were amplified for 35 cycles of 95°C for 20 s, 60°C for 20 s, 72°C for 3 min. Primer pairs used in amplification are listed in Table S4.

Microdissection of sex chromosomes was performed according to described protocol [27 (link)] with some modifications. For each DNA probe, 15–20 copies of the chromosome or chromosome region were collected by extended glass needle using a micromanipulator MR (Zeiss, Oberkochen, Germany). Proteinase K treatment and low temperature cycles of degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) with Sequenase 2.0 (ThermoFisher Scientific, Waltham, MA, USA) were performed exactly as described [27 (link)]. High temperature cycles of DNA amplification were carried out with Encyclo Polymerase mix (Evrogen, Moscow, Russia) in 33 cycles of PCR according to the producer’s recommendation. DNA probes were labeled in an additional 25 cycles of PCR with Tamra-5-dUTP and Fluorescein-12-dUTP (Biosan, Novosibirsk, Russia) as described [28 (link)].

5′-RACE (rapid amplification of cDNA 5′-end) is a method that allows amplification of an unknown sequence on the 5′-end of a specific mRNA, using a primer designed to anneal to a known mRNA part and a universal adaptor primer. RNA extracted from the CNS of a single naive snail was used in this experiment. We used the SMART approach [48 (link),49 (link)] to prepare first-strand cDNA and anchor it with the adaptor sequence in a single step. cDNA synthesis was followed by Step-Out RACE procedure (a specific variant of nested PCR) [50 (link)] to amplify the 5′-end of the sequence. Reverse transcription paired with flanking of cDNA with adaptor sequences was performed with Mint cDNA synthesis kit (Evrogen, Moscow, Russia) according to the manufacturer’s protocol. cDNA was pre-amplified with a single adaptor-specific primer according to Evrogen’s protocol before being used as a template for nested PCR. Three rounds of nested PCR were performed with Mint RACE primer set and Encyclo polymerase mix (Evrogen, Moscow, Russia) using PCR programs recommended by Evrogen. Primer annealing temperature was 62 °C in every reaction. PCR products were visualized using 1% agarose gel electrophoresis.