Rna prep pure tissue kit

Total RNA was extracted using an RNA prep Pure Tissue Kit (Nanjing Vazyme Biotech, Nanjing, China). First-strand cDNA was synthesized using a reverse transcription system kit (Nanjing Vazyme Biotech). The full coding sequence of the non-membrane region of EgHCDH was amplified using the primers 5′- CGG GAT CCA TGT CAG CCG GTG CTG G-3′ (BamHI) and 5′-GAC GTC GAC TCA CTG TTT TTC CTT GAC AAT GCG C-3′ (SalI). Amplification reactions were performed using the following cycling conditions: pre-denaturation at 95 °C, 5 min; then denaturation at 95 °C/30 s, 62 °C/30 s, 72 °C/1 min; with a final extension at 72 °C, 5 min. Through sequencing, digestion and ligation, EgHCDH was ligated into the pET32a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into Escherichia coli BL21 (DE3) cells (Tiangen, Beijing, China). Protein expression was induced with 1 mM isopropyl-1-thio-β-d-galactopyranoside at 37 °C for 6 h. The rEgHCDH protein was purified using Ni²⁺ affinity chromatography (Bio-Rad, Hercules, CA, USA), with the the purity of the final product determined by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The concentrations of the purified protein were determined using a NanoDrop 2000c spectrophotometer (Bio-Rad).

An RNA-prep Pure Tissue Kit (Nanjing Vazyme Biotech, Nanjing, China) was used to extract total RNA from the PSCs. First-strand cDNA was synthesized from total RNA using a reverse transcription system kit (Nanjing Vazyme Biotech). PCR was then used to amplify the LEL coding sequence from the cDNA using a sense primer (5′- CGC GGA TCC ATG TTT CCA GCA CCG CTT CAA G-3′) comprising a BamHI site (underlined) and an antisense primer (5′-CCG CTC GAG TCA TTC ATA GTT TTT CAA GGA G-3′) comprising a XhoL I site (underlined). The PCR amplicons were ligated into the pET32a (+) plasmid (Novagen, Darmstadt, Germany) and transformed into Escherichia coli BL21 (DE3) cells (Tiangen, Beijing, China). Isopropyl-1-thio-β-D-galactopyranoside (1 mM; IPTG) was used to induce expression from the plasmid for 6 h at 37°C. Inclusion bodies were obtained from the E. coli cells, suspended in lysis buffer containing 8 M urea, and incubated for 2.5 h on ice to completely solubilize the recombinant protein. Ni²⁺ affinity chromatography with a His-affinity resin column (Bio-Rad, Hercules, CA, United States) was used to purify the His-tagged rEg-TSP11 protein, which was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A NanoDrop 2000c (Bio-Rad) instrument was used to determine protein concentration.