Sequencing Variable Regions of IgG Antibody

The messenger ribonucleic acid (mRNA) encoding for the variable regions of E10E-1-10 was sequenced by using the template-switch specialized reverse transcription-PCR (RT-PCR) [32 (link)]. Briefly, total mRNA was extracted from the freshly harvested hybridoma cells by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. To synthesize sequence-labeled cDNA, SMARTScribe Reverse Transcriptase kit (Clontech, Mountain View, CA, USA), custom-sequence template-switch oligonucleotide (TSO), and sequence-specific primers for the kappa chain (mIgK RT), lambda chain (mIgL RT), and heavy chain (mIgHG RT) of murine IgG were used (Table 1). For each reaction, 100 ng mRNA, 1 μL of 10 mM primer (mIgK RT, mIgL RT, or mIgHG RT), and 1 μL of 10 mM dNTP were mixed and incubated at 72 °C for 3 min. Subsequently, 2 μL of 5x SMARTScribe buffer (Clontech), 1 μL of 20 mM DTT (Clontech), 3 μL of 10 μM TSO, 0.25 μL of RNAase inhibitor (Roche, Basel, Switzerland), and 0.5 μL of 100 U/μL SMARTScribe Reverse Transcriptase (Clontech) were added to the mixture and the reaction was filled to 20 μL with RNAse-free water. The mixture was incubated at 42 °C for 60 min following a 5-min incubation at 70 °C. The cDNA was stored at −20 °C until use.