Ago HITS-CLIP in PEL cells.

<p><b>A</b>: Western blot (WB) analysis of Argonaute immunoprecipitation with the anti-Ago antibody 2A8 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002884#ppat.1002884-Nelson1" target="_blank">[86]</a> or an unspecific antibody (IgG) as control. The IP was blotted with the anti-Ago2 11A9 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002884#ppat.1002884-Rudel1" target="_blank">[88]</a>. <b>B</b>: Autoradiogram of the radio-labeled RNA in cross-linked, immunoprecipitated Ago-miRNA-mRNA complexes after high or low RNAse treatment. Low RNase treatment yields a 110 and a 130 kDa complex. No complexes are visible with the control IgG. <b>C</b>: PCR products of the 1^st PCR amplification step separated by urea PAGE. Linker-ligated and reverse transcribed RNA extracted from the low RNAse treatment 110 kDa complex (B, lane 3) contained predominantly miRNAs (20–25 nt+∼40 nt linkers). The 130 kDa complex contained miRNAs and 50–70 nt mRNA tags (90–110 nt including linkers). MiRNA and mRNA PCR products of the 130 kDa complex shown in lane 1 were extracted for the 2^nd PCR amplification. <b>D</b>: products of the 2^nd PCR separated on an agarose gel. The longer primers used in this PCR amplification step yielded ∼115 bp products for the miRNAs and 140–160 bp products for the mRNAs. <b>E</b>: Scatter plot showing the correlation of the miRNA CLIP tags between biological replicates (BR) in BCBL-1 (shown as log₂ of the miRNA frequency; for reproducibility in BC-3 see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002884#ppat.1002884.s005" target="_blank">Figure S1B</a>). <b>F</b>: distribution of mRNA-annotated clusters across transcripts. <b>G</b>: differential recovery of KSHV and human miRNAs associated with Ago in BCBL-1 and BC-3.