(a) Schematic of hORFeome V8.1 creation. Templates from the Mammalian Gene Collection (MGC) were transferred into the Gateway system via PCR and recombinational cloning, resolved as clonal isolates, fully sequenced and rearrayed. (b) Sequencing outcomes for 19,281 ORF samples in polyclonal format, from which single colonies were isolated. 14,524 ORFs were fully sequenced and accepted into the final collection (Complete, accepted). 198 ORFs were fully sequenced, but rejected for lacking a start codon (Complete, rejected). 825 ORFs were partially sequenced, including undetermined nucleotides (Partial). 823 ORFs were made clonal but were intentionally not sequenced, since these ORFs were isoforms of other ORFs in the sequencing pool and could cause unambiguous read mapping (Not attempted). See Supplementary Figure 3 for more details. (c) Alignment of the 14,524 completely sequenced clones with MGC templates. 12,736 clones have identical sequence as templates or have one synonymous error only (Perfect), and another 1,788 clones have additional mutations (Mutant). (d) Alignment of the 14,524 completely sequenced clones with NCBI RefSeq transcripts. 10,216 ORFs represent full length coding sequences with > 99% homology (Full), 1,545 ORFs were partial length coding sequences with > 85% homology (Partial) and 2,763 clones fell into other categories (Other). See Supplementary Figure 4 for more details.

(a) Schematic of the creation of the CCSB-Broad Lentiviral Expression Library. pLX304-Blast-V5 is a custom lentiviral vector validated for high-throughput screening encoding Blasticidin (Blast) resistance and a C-terminal V5-epitope tag. (b) Evaluation of high-throughput ORF transduction and expression in A549 lung cancer cell lines. The micrographs show images of cells stained for cellular DNA (to assess cell number) and with antibodies recognizing the V5 epitope (to assess ORF expression) after lentiviral infection and three days of growth in blasticidin. Wells in which no virus was added are highlighted with yellow outline. (c) Distribution of ORF sizes and average viral titer as a function of ORF size. (d) ORF expression as a function of ORF size. ORFs larger than 3 kb showed a decreased yet detectable above-background level of expression. Background was assessed from cells expressing a control vector without V5 expression.