Saccharomyces cerevisiae eby100 cells

Saccharomyces cerevisiae YVH10 cells (ATCC) were used in antibody library generation and FACS sort. Hela-hACE2 cells (Rogers et al., 2020 (link)) were used in pseudovirus neutralization assay. Saccharomyces cerevisiae EBY100 cells (ATCC) were used in RBD mutant library generation and FACS sort. Human HEK293T cells (ATCC) were used for pseudovirus production. FreeStyle HEK293 cells (ThermoFisher) were used for recombinant S protein production. Expi293F cells (ThermoFisher) were used for monoclonal antibody and recombinant RBD production. Vero-E6 cells (ATCC) were used for live virus plaque assay.

Nymphal I. ricinus ticks were fed on rabbits for 24 (800 nymphs), 48 (400 nymphs) and 72 h (150 nymphs). After removal from the rabbit, salivary glands were isolated, pooled, lysed in ML buffer (MACHEREY–NAGEL GmbH & Co KG) and stored at − 80 °C. Small and large RNA was extracted using the miRNA kit (MACHEREY–NAGEL GmbH & Co KG) for each time point and pooled. cDNAs were prepared and directionally cloned into the EcoRI and NotI sites of the yeast expression vector pYD1 (Invitrogen, CA). The subsequent cDNA library was normalized to Cot40. PstI digestion of plasmids of the pYD1-salivary gland library, showed an average insert size of 1.5 kb and 100% of the clones contained inserts. The unamplified library titre was 2,8 × 10⁶ cfu/ml. Yeast cells were transformed by electroporation as described by Chao et al⁵⁶ . Briefly, fresh Saccharomyces cerevisiae EBY100 cells (ATCC, Manassas, Virginia, USA) were electroporated together with 5 µg of DNA and subsequently grown in SDCAA medium (2% dextrose, 0.67% yeast nitrogen base, 0.5% bacto amino acids, 30 mM NaHPO4, 62 mM NaH2PO4) at 30 °C.