Construction of IVT-seq libraries. (A) Preparation of a pool of 1,062 human cDNA plasmids. Contents of three 384-well plates containing MGC plasmids were pooled together. Pool was amplified via transformation in Escherichia coli, and resulting clones were purified and re-pooled. (B) Generation of IVT transcripts. Pool of MGC plasmids was linearized and used as a template for an in vitro transcription reaction. Enzymes and unincorporated nucleotides were purified, leaving pool of polyA transcripts. (C) Creation of IVT-seq libraries. Listed quantities of IVT RNA were mixed with mouse liver total RNA to create six pools with final RNA quantities of 1 μg. Ribosomal RNA was depleted from these pools using the Ribo-Zero Gold kit. IVT RNA and mouse RNA are now present in pools at the listed ratios, following depletion of rRNA from mouse total RNA. These pools were used to generate RNA-seq libraries using Illumina’s TruSeq kit/protocol. This entire process was performed in duplicate. Replicate libraries were pooled separately and sequenced in separate HiSeq 2000 lanes (two lanes total). IVT, in vitro transcribed; MGC, Mammalian Gene Collection.