Extended Data Fig. 5: PAR-CLIP experiments identified viral and host targets of miR-138.
From: Regulation of host and virus genes by neuronal miR-138 favours herpes simplex virus 1 latency
a, miR-138 expression in 293 Tcontrol and 293T138 cells. b, PAR-CLIP procedure for detecting viral targets of miR-138 with, from left to right, a schematic showing the samples, a cartoon showing the crosslinking procedure, a representative autoradiograph of an SDS polyacrylamide gel showing a band corresponding to the Ago-RNA complex, the subsequent steps, and an agarose gel showing PCR amplification of the cDNA library. The experiment was performed once. c, Read counts of the indicated sequences from the indicated samples. d, 293T cells were co-transfected with 20 nM miRNA mimic and 100 ng/ml plasmid for 48 h before Western blot analysis of FLAG-tagged UL39 using an anti-FLAG antibody and analysis of ICP0 using an ICP0 antibody. This experiment was repeated once with similar results. e, miR-138 levels in Neuro-2a, N2A138 and N2Aanti138 cells. f, Diagram showing the “anti138” sequence expressed in N2Aanti138 cells. The sequence has a “tough decoy” secondary structure. Red and black horizontal lines represent anti138 and miR-138, respectively. Curved lines above and below the main structure represent bulges (extra nucleotides not bound to miR-138) designed to prevent cleavage. g, miR-138 and total read counts in the N2A138 and N2Aanti138 cells in a PAR-CLIP experiment. h, Fraction of 5’ UTR, CDS or 3’ UTR sites in total sites identified by the single PAR-CLIP approach (panels 1 and 2) or the combined PAR-CLIP/RNAseq approach (panels 3 and 4) in 293 T (panels 1 and 3) and Neuro-2a cells (panels 2 and 4). Relative to the single PAR-CLIP approach, the combined approach identified significantly higher fractions of targets with 3’ UTR sites (P = 0.034 and 0.0006 for 293 T and Neuro-2a cells, respectively by Fisher’s exact tests). For a and e, n = 3 biologically independent samples and data are presented as mean values ± s.d.
