Fig. 1: A massive parallel 3’-UTR reporter assay.

a The pRPa^iUTR reporter construct. A blasticidin S-deaminase (BSD) and thymidine kinase (TK) fusion gene, with positive or negative regulatory 3’-UTRs cloned immediately downstream of the stop codon, was placed under the control of a tetracycline-inducible rDNA promoter and flanked by homology regions (X1 is HYG^Δ and X2 is rDNA) to integrate the full construct at the tagged rDNA spacer locus in the 2T1 T. brucei strain; both RNA polymerase I and RNA polymerase II are used to drive protein coding gene transcription in T. brucei. A constitutively expressed NPT cassette under the control of a bloodstream VSG expression site (ES) promoter was also included to allow selection of recombinants in the absence of tetracycline. b Dose response curves reveal relative blasticidin (BSD) resistance and ganciclovir (GCV) sensitivity when reporter expression is increased (green); see Supplementary Fig. 1a. c The cassette immediately downstream of the BSD-TK stop-codon facilitated high-efficiency library construction. pRPa^iUTR was digested with BbsI and T semi-filled, while T. brucei genomic DNA was partially digested with Sau3AI and fragments of 1–3 kbp were G semi-filled prior to ligation. The FseI sites and index sequences facilitated assessment of library complexity and fragment orientation. d The massive parallel reporter assay. The plasmid library was used to assemble a T. brucei library, which was induced with tetracycline, selected with BSD or GCV, and subjected to UTR-seq. e We sampled the library at days 4, 6 and 8 (lighter to darker shading), extracted genomic DNA, amplified cloned library fragments by PCR, deep-sequenced the products, mapped reads to annotated 3’-UTRs, and compared the outputs using principal component analysis. f Mapped reads adjacent to 48,509 Sau3AI sites in the T. brucei genome were quantified. Data for the plasmid library and sequences recovered following 6 days of selection are shown.