NGS of GPDmuc-foldon_additional_run2_M3: antibody heavy chain library
Briefly, 5-RACE cDNA was obtained from bulk-sorted splenic B cells of each mouse with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (TaKaRa). The IgG PCRs were set up with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a total volume of 50 l, with 5 l of cDNA as the template, 1 l of 5-RACE primer, and 1 l of 10 M reverse primer. The 5-RACE primer contained a PGM/S5 P1 adaptor, while the reverse primer contained a PGM/S5 A adaptor. We adapted the mouse 3-Cg1-3/3-Cm inner primers and 3-mCk outer primer as reverse primers for the 5-RACE PCR processing of heavy and light (k) chains. A total of 25 cycles of PCR was performed and the expected PCR products (500-600 bp) were gel purified (Qiagen). NGS was performed on the Ion S5 GeneStudio system. Briefly, heavy and light (k) chain libraries from the same mouse were quantitated using a Qubit 2.0 Fluorometer with Qubit dsDNA HS Assay Kit and then mixed at a 3:1 ratio before being pooled with antibody libraries of other mice at an equal ratio for sequencing. Template preparation and (Ion 530) chip loading were performed on Ion Chef using the Ion 520/530 Ext Kit, followed by sequencing on the Ion S5 system with default settings.
SUB9396384
GPDmuc-foldon_additional_run2_M3