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. 2020 Jun 5;48(13):7439–7453. doi: 10.1093/nar/gkaa478

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — Characterization of CRISPR-derived knockout cell lines. (A–C) Location of guide RNAs and mutations knocking out AGO1, AGO2 and AGO3 expression. (D) Western blot validating the AGO1, AGO2, AGO1/2 and AGO1/2/3 knockout cell lines (n = 3). (E) Effect of knockouts on growth rates. WT, AGO1, AGO2, AGO1/2 and AGO1/2/3 knockout cell lines were seeded at the same density, harvested, and analyzed for live cell count (n = 3). A Two-Way ANOVA with Bonferroni post-test analysis show that the AGO2−/− (P value 0.0066), AGO1/2−/− (P value 0.0045), and AGO1/2/3−/− (P value 0.0002) growth rates are significantly different from WT. (F) Scheme showing knockout of AGO variants, RNAseq analysis of gene expression changes relative to wild-type cells; and eCLIP-sequencing with anti-AGO2 antibody to identify potential miRNA–AGO2 binding sites. See also Supplemental Figure S1.