Rnaase inhibitor

For reverse transcription, 200-300ng of total RNA was used. The reaction was performed using MultiScribe reverse transcriptase (Thermo Fisher Scientific), random hexamers and RNAase inhibitor (both Roche). Subsequently, the qPCR reaction was performed using the SYBR green GoTaq qPCR master mix (Promega) on Agilent Stratagene Mx3005P qPCR instrument. Sequences of primers are listed in Supplementary Table 1. Relative gene expression was calculated using the 2^-ΔΔCt method. GAPDH was used as the reference gene.

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.