Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to metabolic stress

Chad N. Brocker; Donghwan Kim; Tisha Melia; Kritika Karri; Thomas J. Velenosi; Shogo Takahashi; Jessica A. Bonzo; David J. Waxman; Frank J. Gonzalez

Chromatin was prepared from hepatocytes for ChIP assays as previously described (Kim et al., 2014) . Cells were fixed with 4% paraformaldehyde for 15 min, then glycine was added to a final concentration of 0.125 M and incubated for 10 min before harvesting. Chromatin was sonicated using a Bioruptor Pico (Diagenode, Denville, NJ, USA). Chromatin preparations were subjected to ChIP using a ChIP-IT High Sensitivity Kit and Protein G Agarose Prepacked Columns (Active . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/675785 doi: bioRxiv preprint Motif, Carlsbad, CA, USA) using either PPARA (Abcam; Ab24509) antibody. Normal rabbit IgG (Cell Signaling Technologies; #2729S) and Histone H3 (Cell Signaling Technologies; #4620) antibody were used as negative and positive controls, respectively. DNA was purified and concentrated using MinElute Reaction Cleanup columns (Qiagen). qRT-PCR and conventional PCR were performed using 2 µl of ChIP DNA samples from the 50 µl of purified samples using gene-specific primers (Table S4) . Cycle threshold (Ct) values of ChIP and input samples were calculated and presented as fold change.