Bulk RNA-sequencing of defined brain regions of mice with chronic DSS-induced colitis and colitis induced bz intestinal epithelial-specific Opa1 deletion
Bulk RNA-sequencing of defined brain regions of mice with chronic DSS-induced colitis and colitis induced bz intestinal epithelial-specific Opa1 deletion
For the cDSS samples, mice were sacrificed on the fourth day after the last DSS cycle and transcardially perfused. For the Opa1 samples, on day 24 after first tamoxifen administration, mice were sacrificed, followed by transcardial perfusion. From the right brain hemisphere of all samples, the amygdala, the hippocampus, the hypothalamus and the insular cortex were micro-dissected, RNA was isolated and sent to Novogene GmbH, Munich, Germany for quality control, library preparation and bulk RNA sequencing. For the chronic DSS samples, male C57BL/6J mice received three cycles (4-5 days each) of 1.5% DSS in their drinking water followed by a 2-week recovery phase with regular drinking water. Control mice received normal drinking water throughout the experiment. For the Opa1dIEC mice, Opa1fl/fl x VillinCreERT2 mice (Bao et al., Sci Immunol (2025)) were treated with 75 mg/kg body weight tamoxifen p.o. for five consecutive days to induce Opa1 deletion specifically from the intestinal epithelium leading to epithelial barrier damage and colitis. RNA isolation was performed using the RNeasy Mini kit (QIAGEN) following the manufacturerâ€™s instructions. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.