Figure 1. Modular Barcoded Human ORF Libraries and Inducible Expression System.
(A) Construction of ORF library expression vector. Libraries of random oligos (BC Library) flanked by primer landing sites were cloned into the vector using rare unique restriction sites I-CeuI and I-SceI. ORF collections were cloned into Gateway DEST site by LR recombination. The libraries were then sheared and resulting ORF-BC pairs were recovered by PCR and identified by paired-end sequencing. LTR, long terminal repeat; TRE, tetracycline responsive element; DEST, Gateway Destination cassette; attB1/2, Gateway recombination sites; PGK, phosphoglycerate kinase 1 promoter; Puro, puromycin resistance gene.
(B) Maps of two-component system for inducible expression of barcoded ORFs. ORFs are expressed from pHAGE-TRE-ORF-PGK puro-3′BC library vector under control of the reverse tetracycline transactivator (rtTA), which is expressed from pInducer-rtTA-Neo. Ubc, ubiquitin C promoter; IRES, internal ribosome entry site; Neo, neomycin resistance gene.
(C) Flow cytometry measurement of induction of GFP expressed from pHAGE-TRE-ORF-PGKPuro-3′BC in either a heterogeneously infected population of rtTA-Neo expressing HMECs or a clonal rtTA-HMEC line (Clone 1-9). Cells were induced with 100 ng/mL dox for 48 hr before analysis or left untreated.
(D) Western blot for GFP expression at indicated dox concentrations (in ng/mL) in parental rtTA-HMEC population and rtTA-HMEC Clone 1-9. GAPDH is used as a loading control.
(E) Distribution of the frequency of ORFs paired to a given number of unique BCs in each of the ORF libraries.