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. 2017 Nov 9;7:15178. doi: 10.1038/s41598-017-15388-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Characterization of the miRNome differentially associated with the AGO-2 complex in response to IL-1β treatment in primary human chondrocytes. Primary human chondrocytes were left untreated or treated with IL-1β (5 ng/ml) for 16 hrs followed by RNA immunoprecipitation using AGO-2 antibody (AGO2-RIP). The eluted RNA was used for library preparation using small RNA library preparation kit (Illumina) and next generation sequencing on Illumina platform. 10% input RNA was sequenced in parallel. Data were analyzed using Illumina small RNA app available on BaseSpace. (A) M-A plot showing the differential abundance of the miRNAs/isomiRs associated with AGO2 (blue dots) compared to input RNA (red dots). (B) Volcano plot showing the q Value of differentially expressed miRNAs/isomiRs vs Log2 Fold change. (C) Western blot analysis showing the expression and phosphorylation of the AGO2 protein in response to IL-1β. (D) Fold change in the expression of 6 top ranking miRNAs/isomiRs as per the data obtained by NGS. (E) The NGS data shown in D were validated by TaqMan real time PCR using the primers available for canonical sequences only. (F) Enrichment of the selected targets of miR-146a-5p and miR-155-5p in the AGO2 fractions prepared from control cells and IL-1β treated cells was quantified by real time analysis. Data are presented as relative fold enrichment of the selected mRNAs in the AGO2 fraction isolated from IL-1β-treated chondrocytes compared to untreated chondrocytes.