Mint kit

Sequencing of mRNAs encoding heavy and light chains of antibodies was performed using 5′ SMART RACE method as described previously [49 (link)] with slight modifications. The first strand of cDNA was synthesized using Mint kit (Evrogen, Moscow, Russia) with primers for k-chain (TTG TCG TTC ACT GCC ATC AAT C), λ-chain (GGG GTA CCA TCT ACC TTC CAG), and heavy-chain (CTG GAC AGG GAT CCA GAG TTC CA) according to the manufacturer’s protocol. Next, cDNA encoding immunoglobulins was amplified by conventional PCR using M1 forward primer (AAG CAG TGG TAT CAA CGC AGA GT) and revers primers specific for k-chain (ACA TTG ATG TCT TTG GGG TAG AAG), λ-chain (ATC GTA CAC ACC AGT GTG GC), and heavy-chain (GGG ATC CAG AGT TCC AGG TC). Obtained DNA was purified and cloned into pKAN-T (Evrogen, Moscow, Russia) vector that was subsequently sequenced. At least 3 different clones were sequenced for each chain of the antibodies. Immunoglobulin sequences were analyzed using IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/), BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE), SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/) and Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) software.

cDNA was obtained from total RNA using the Mint kit (Evrogen, Moscow, Russia) according to the manufacturer’s protocol. For PC3 cell samples, qPCR was performed on an Applied Biosystems 7500 instrument (Thermo Fisher Scientific, USA) in three technical replicates. HPRT1 was used as a control gene. For blood plasma exosome samples, qPCR was performed on a Rotor-Gene Q instrument (Qiagen) in three technical replicates. GAPDH was used as a control gene. The level of relative mRNA expression for each comparison was calculated by the ΔCT method. Visualization and statistical analysis of expression results were performed using t and paired Wilcoxon tests in Jupyter Notebook, Python (ver. 3.6, Python Wilmington, DE, USA).

Total RNA was extracted from frozen cell pellets with TRIzol™ Reagent (Life Technologies, Carlsbad, CA, USA) and the following chloroform phase separation and ethanol precipitation at −20 °C. Washed RNA was dissolved in Steril RNAse-free water (Thermo Fisher Scientific, Waltham, MA, USA). The final RNA concentration was measured using a spectrophotometer. To prevent RNA aggregation, total RNA samples were heated at 65 °C for 1–2 min before cDNA synthesis; samples were immediately used in cDNA synthesis. DNAse treatment was performed with RQ1 RNAse-free DNAse (Promega, Madison, WI, M6101). First-strand cDNA was synthesized from 1 µg total RNA using Mint Kit (Evrogen, Moscow, Russia) according to the manufacturer’s instructions.

The same strain was used to generate a normalized EST library. The fungus was pre-cultivated in 50 ml of 2% malt broth (20 g l^-1 malt extract; Difco) for 14 days at 20 °C under constant shaking. Then, the mycelium was homogenized with a blender for 30 s and 5 ml of the homogenized mycelium was transferred to new 50 ml of 2% malt broth (20 g l^-1 malt extract; Hefe Schweiz). After 48 h the actively growing mycelium was harvested and immediately frozen in liquid nitrogen. Total RNA was isolated from approx. 75 mg fresh mycelium using the RNeasy plant mini kit (Qiagen, Hombrechtikon, Switzerland). Full-length cDNA was synthesized using the MINT kit (evrogen, Moscow, Russia) with a degenerated poly-T primer (5′-AAGCAGTGGTATCAACGCAGAGTAC (T)₄G(T)₉C(T)₁₀VN-3′) during the first strand cDNA synthesis [95 (link)] and polTM1 (5′-AAGCAGTGGTATCAACGCAGAGTACTTTTGTCTTTTGTTCTGTTTCTTTTVN-3′) for the generation of dsDNA. The cDNA was normalized using the TRIMMER kit (evrogen) and the library was sequenced on the 454. Resulting reads were filtered for chimeras and then a whole transcriptome assembly was performed in newbler 2.3 with a minimum overlap of 50 bases and 98% sequence similarity.

One microgram of total RNA obtained by combining RNA preparations from all samples were used for the rapid amplification of cDNA ends using the Mint kit (Evrogen, Moscow, Russia) according to the manufacturer’s instructions. The amplified cDNA coding SlHev1 was synthesized using gene-specific primers (Supplementary Materials Table S4) constructed using the Beacon Designer 4.0 program and high-fidelity Tersus DNA polymerase (Evrogen, Moscow, Russia). PCR conditions were as follows: initial denaturation step at 94 °C for 2 min followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 58 °C for 30 s, and primer extension at 72 °C for 30 s, with the final extension of 5 min at 72 °C. The amplified fragment was separated by agarose gel electrophoresis and isolated from the gel with the Cleanup Standard kit (Evrogen, Moscow, Russia). The PCR fragment was cloned into the pAL2-T vector (Evrogen, Moscow, Russia). The construct obtained was sequenced on an ABI PRISM 3730 instrument (Applied Biosystems, Foster City, CA, USA).

Total RNA was purified from the venom glands of T. oblongus using Trisol® Reagent (Ambion, Canada) according to manufacturer protocol. Total cDNA was synthesized from 5 μg of total RNA, using the MINT kit (Evrogen, Russia) following the manufacturer recommendations. Rapid amplification of cDNA ends was carried out using the universal primer T7cap (GTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA ACA ACG CAG AGT) and degenerated primers To1 (TGT GCC AGC AAG AAY GAR MGN TGY GGN AAY) and To2 (GCG AGC AAG AAT GAR MGN TGY GGN AAY GCN) for 3′-terminus determination (3′-RACE) and To3 (ACG CGC AGT TTC TTR CTY KCN ACR CCN) for 5′-terminus determination (5′-RACE). DNA sequencing was carried out on ABI PRISM 3100-Avant.

Total RNA was extracted from defrosted and homogenized SH-SY5Y cells with TRIzol™ Reagent (Life Technologies, Carlsbad, CA, USA) and the following chloroform phase separation and ethanol precipitation at −20 °C. Washed RNA was dissolved in Steril RNAse-free water (Thermo Fisher Scientific, USA). Final RNA concentration was measured using a spectrophotometer. To prevent RNA aggregation, total RNA samples were heated at 65 °C for 1–2 min before cDNA synthesis. First-strand cDNA synthesis was performed using Mint Kit (Evrogen, Moscow, Russia) according to the manufacturer’s protocol (1.5 μg RNA for each reaction).