Experimental scheme for AGO2-eCLIP-sequencing. (A) Scheme showing how knockout of AGO variants, eCLIP, and nuclear RNA-seq analysis of alternative splicing relative to wild-type cells are used to identify and validate candidate genes. Wild-type and HCT116 knockout cells were used for RNA-seq. Wild-type and AGO2 knockout cells were used for eCLIP. (B) Microscopic imaging to evaluate the association of endoplasmic reticulum (ER) with purified nuclei. (Left) Nuclei after one-time wash with hypotonic buffer containing 0.5% NP40. (Right) Nuclei after seven times wash with hypotonic buffer containing 2.0% NP40. (Blue) Nuclear staining with DAPI; (red) endoplasmic reticulum marker. (C) Western blot of AGO1, AGO2, AGO1/2, and AGO1/2/3 knockout cell lines to evaluate the purity of nuclear and cytoplasmic samples and presence of AGO protein variants (representative of n = 3). Quality was confirmed for all samples prior to submission for RNA-seq.
