Single-cell ORF perturbation screening of an enriched viral microprotein library in hTERT RPE-1 cells using 10x Genomics Flex fixed-RNA profiling

To assay their cellular consequences at scale, we assembled an enriched viral microprotein ORF library in the doxycycline-inducible pANDORA lentiviral vector and delivered it to hTERT RPE-1 cells expressing the pINDUCER20 at low multiplicity of infection (<30% infection rate). The library was split into two sublibraries by insert length ("300mer" and "400mer").

Transduced cells were induced with doxycycline and collected at two time points (24 h and 48 h post-induction), then fixed and profiled by single-cell transcriptomics using the 10x Genomics Single Cell Gene Expression Flex (fixed RNA profiling) assay with 16-plex probe barcoding. ORF identity was read out per cell from a custom probe panel targeting the library inserts, amplified into a dedicated ORF feature library. This dataset comprises paired whole-transcriptome and ORF-assignment measurements for individual cells, enabling the transcriptional consequence of each viral microprotein to be resolved against isogenic controls in the same pool.