Ultralong Cow Antibodies
Bovine spleen and lymph nodes were obtained from Animal Technologies (Tyler, TX), or from Texas A&M University. Total RNA was isolated from bovine tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol, followed by on column digestion of DNA using the RNeasy Mini Kit (QIAGEN, Valencia, CA). RNA quantity and quality were assessed with Nanodrop (Thermal Scientiﬁc), Qubit RNA, and Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA), following the manufacturer’s protocols. Total RNA was used as a template for complementary DNA (cDNA) synthesis catalyzed by Superscript II (Invitrogen). The antibody variable region was ampliﬁed from cDNA using primers 5'-TTGAGCGACAAGGCTGTAGGCTG-3' and 5'-CTTTCGGGGCTGTGGTGGAGGC-3'. Bar-coded primers (Table S2) were used to amplify VH from bovine spleen cDNA. The amplicons of VH were puriﬁed from 2% agarose gels and deep sequenced according to Roche 454 GS FLX instructions.