Cel mir 39 1

Six boar SP-EVs miRNAs were randomly selected (four higher sequence reads miRNAs: ssc-miR-21-5p, ssc-miR-148a-3p, ssc-miR-10a-5p, and ssc-miR-125b, and two lower sequence reads miRNAs: ssc-miR-135 and ssc-miR-744). Reverse transcription was performed according to the TransScript miRNA First-Strand cDNA Synthesis SuperMix kit (Transgen, Cat. No. AT351-01). These cDNAs were validated by SYBR-Green qPCR using PerfectStartTM Green qPCR SuperMix kit (Transgen, Cat. No. AQ602-21) on a Real-Time PCR System (Applied Biosystems). The spike-in control cel-miR 39-1 (Qiagen, Cat. No. 219610) was used as endogenous control. Universal reverse primer for qPCR was obtained from TransScript miRNA First-Strand cDNA Synthesis SuperMix kit (Transgen, Cat. No. AT351-01). All measurements were analyzed in another six boar SP-EVs samples (n = 6). Relative miRNA expression was calculated using the 2^−−ΔΔCt method (32 (link)).

The RNA samples from three cyclic and three pregnant UFs’ EVs were analyzed by quantitative PCR (RT-qPCR). Six differentially expressed miRNAs were randomly selected (chi-miR-140-5p, chi-miR-10b-5p, chi-miR-127-3p, chi-miR-17-5p, chi-miR-200a, chi-miR-30a-5p). Reverse transcription was performed with the Mir-X miRNA First-Strand Synthesis Kit (Takara, Cat. No. 638315), according to the manufacturer’s protocols. Subsequently, these cDNAs were validated by qPCR using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher, Cat. No. A25742) on a Real-Time PCR System (Applied Biosystems). The spike-in control cel-miR 39-1 (Qiagen, Cat. No. 219610) was used as the endogenous control. The forward primer sequences are visible in Supplementary Table S2, while the reverse primer was obtained from the above kit. The efficiencies of the amplification curves and the quantification cycle (Cq) were determined using LinRegPCR software [46 (link)], and then the relative expression levels of miRNA were calculated using the 2^−△△Ct method [47 (link)].