Transscript 2 all in one first strand cdna synthesis supermix for pcr

All H. flava samples were collected from the body surface of hedgehogs in the city of Xinyang in Henan Province (31°53′N, 114°04′E), China, and identified using a stereomicroscope based on previously described morphological characteristics [26 ]. The surfaces of the ticks were cleaned and sterilized with 75% ethanol. Total RNA was extracted using an EasyPure RNA Kit (TransGen Biotech, Beijing, China). The quality of RNA was assessed by electrophoresis in a 1% agarose gel. RNA samples with clear and distinct electrophoretic bands as a template were selected for synthesizing H. flava complementary DNA (cDNA) using TransScript® II All-in-One First-Strand cDNA Synthesis SuperMix for PCR (TransGen Biotech).

Results of RNA-seq were validated via RT-qPCR experiments. Real-time PCR analyses were performed as the user manual of the TransScript II All-in-One First-Strand cDNA Synthesis SuperMix for PCR (TransGen Biotech) and the SYBR Premix Ex Taq II kit (Roche) described. The housekeeping gene was GhActin. The gene-specific primers designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) tool and primers are listed in Additional file 5: Table S1, Additional file 6: Table S2, Additional file 7: Table S3, Additional file 8: Table S4, Additional file 9: Table S5, Additional file 10: Table S6, Additional file 11: Table S7, Additional file 12: Table S8. The experiments of real-time PCR were performed using three biological replicates for each tissue sample and at least three technical replicates of each biological replicate. The value of genes folds change was calculated using the 2^-ΔΔCT method [65 ].

Fab phage display libraries were constructed using the pComb3X vector expression system based on a well-established protocol (Protocol 9.1: Human Fab Libraries)[24 ]. Peripheral blood mononuclear cells (PBMC) were isolated from fresh whole blood within 2 h after collection using the Lymphocyte Separation Medium (TBDsciences, Tianjin, China). Total RNA was extracted from PBMC using the EasyPure RNA Kit (Transgen, Beijing, China), and cDNA was synthesized from the total RNA sample using the TransScript II All-in-One First-Strand cDNA Synthesis SuperMix for PCR (Transgen, Beijing, China). To generate Fab gene segments, a three-step PCR method was performed using PrimeSTAR HS DNA Polymerase with GC Buffer (TaKaRa, Tokyo, Japan). The resultant Fab gene segments were digested with restriction enzyme Sfi I (New England Biolabs, Ipswich, MA, USA), and ligated into the phagemid pComb3XSS that had been cut with the same restriction enzyme using T4 DNA Ligase (New England Biolabs, Ipswich, MA, USA). Recombinant plasmids were transformed into competent Escherichia coli (E. coli) TG1 cells by electroporation (Bio-Rad, Hercules, CA, USA).