Taq pcr mastermix

Total mRNA was extracted from the cells using TRIpure Reagent (BioTeke Biotechnology Co., Ltd, Beijing, China). After cDNA synthesis using BeyoRT II M-MLV (Beyotime Biotechnology Co., Ltd, Shanghai, China) based on the instruction, quantitative real-time PCR was carried out using 2×Taq PCR MasterMix (Solarbio Biotechnology Co., Ltd, Beijing, China) and SYBR Green (Solarbio Biotechnology Co., Ltd) on an Exicycler 96 PCR system (BIONEER, Daejeon, Korea). CDNA served as the template. The following primers were used: forward primer for USP44, 5'-CAA CTT ATG ATA TGC CAC CTA-3'; reverse primer for USP44, 5'-GTA CCC AGA ACC CTC CT-3'; forward primer for WDR5, 5'-CAC CTG TGA AGC CAA ACT-3'; reverse primer for WDR5, 5'-GAG GCA GAA ACA AGA AGG-3'; forward primer for GAPDH, 5'-GAC CTG ACC TGC CGT CTA G-3'; reverse primer for GAPDH, 5'-AGG AGT GGG TGT CGC TGT-3'. The thermal circulation is as follows: 94 °C for 5 min; 94 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s, 40 cycles; then 72 °C for 2.5 min and 40 °C for 1.5 min. It was melting form 60 °C to 94 °C, 1 °C/1 s. Gene expression was determined based on the 2^-ΔΔCT method and normalized to GAPDH.

Total RNA was extracted from cells with TRIzol Reagent and from EMNVs with a Total Exosome RNA & Protein Isolation Kit (both from Invitrogen, Carlsbad, CA) referring to the guidebook provided. For mRNA and LncRNA, cDNA was synthesized using a TransScript All-in-One First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China) and PCR was performed using 2 × Taq PCR MasterMix (Solarbio, Beijing, China) according to the steps described in the handbooks.
Primers used in the present study are shown in Table 1.
Further, quantitative PCR (qPCR) was performed following the procedures described previously (Tao et al., 2017d (link)) for precise quantification.

Total RNA isolation was carried out from infected cells by using the TRIpure (BioTeke, Beijing, China) according to the manufacturer’s protocol. Then, the RNA was reverse transcribed into cDNA with the BeyoRT II M-MLV reverse transcriptase (Beyotime, Shanghai, China). Subsequently, amplification was performed by employing the 2 × Taq PCR MasterMix (Solarbio, Shanghai, China) and SYBR Green (Solarbio). The expression of RUNX1 was calculated with 2^−ΔΔCt method. β-actin was employed as the internal control. The sequences of primer were as follows. Forward primer: 5′-GACCCTGCCCATCGCTTTC-3′; Reverse primer: 5′-AATCTCGCCACTTGGTTCTTC-3′.

The DNA was extracted using an GenElute™ Soil DNA Isolation Kit (DNB100, Sigma-Aldrich, China). Afterwards, total DNA was purified and concentrated. The DNA samples were further diluted to 1 ng/μL with sterile water, and were then analyzed by agarose gel electrophoresis and quantified by NanoDrop™ 2000 spectrophotometer. The universal primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 909R (5′-GACTACHVGGGTATCTAATCC-3′) were used to target the V3-V4 hypervariable regions of bacterial 16S rRNA gene^{22 (link),34 (link)}. The PCR amplification was carried out with 2× Taq PCR Master Mix (Solarbio^® LIFE SCIENCES, China). The PCR products were analyzed by electrophoresis using 2% agarose gel. Samples with bright main strip between 400–450 bp were chosen for further experiments. PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA). The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Lastly, the libraries were sequenced by Illumina HiSeq 2500 System.

MIHA (Human normal liver cell), LX-2 (Human normal stellate cell), and four human HCC cell lines, including huh7, HepG2, Li-7, and PLCPRF5, were all purchased from ATCC, and were cultured in DMEM (Gibco) supplements with 10% fetal bovine serum (Hyclone), 100 U/ml of penicillin (Gibco), and 0.1 mg/ml of streptomycin (Gibco) at 37°C in a humidified atmosphere of 95% O₂, 5% CO₂.
Total RNA was isolated using the TRIpure Reagent (RP1001, Bioteke). Reverse transcription was performed on 1 mg of RNA at 60°C for 35 min using a BeyoRT II M-MLV (D7160L, Beyotime). After reverse transcription, the cDNAs were used for semi-quantitative PCR by using 2×Taq PCR MasterMix (PC1150, Solarbio) and SYBR Green (SY1020, Solarbio). Amplification was carried out as recommended by the manufacturer in the qPCR instrument (Exicycler 96, BIONEER): 25 µl of reaction mixture contained 12.5 µl of SYBR Green mastermix, the appropriate primer concentration, and 1 µl of cDNA. The amplification program included the initial denaturation step at 95°C for 10 min, 40 cycles of denaturation at 95°C for 10 s, and annealing and extension at 60°C for 1 min. Fluorescence was measured at the end of each extension step. After amplification, melting curves were acquired and used to determine the specificity of the PCR products. 2 ^-△△CT method was used in data process.
The sets of specific primers as follows: