Antigen identification by cDNA expression library analysis

Rat C6 glioma cells (ATCC, CCL-107) were obtained from ATCC and transduced with a lentiviral library encoding version 5.1 of the human ORFeome library³⁹ and screened by FACS for reactivity against HIC0-3C5. Two cycles of sorting HIC0-3C5⁺ C6 cells, amplification in culture and re-sorting yielded sufficient material for DNA and RNA recovery. PCR amplification of the cDNA insert from HIC0-3C5⁺ C6 cells was unsuccessful, so an RNAseq library was constructed and sequenced. The resulting sequences were searched for reads corresponding to ORFeome integrations and then compared with human cDNA sequences present in ORFeome 5.1 by BLAST²⁷. Of the 17 mappable reads, 14 matched the 5′ region of ST8SIA1. In addition, the expression of ST8SIA1 was found to be consistently enriched in the RNA of FACS-sorted HIC0-3C5⁺ versus HIC0-3C5⁻ specimens by qRT–PCR and RNA-seq.