title: Bulk RNA-Sequencing of brain hemispheres of mice with colitis induced by intestinal epithelial-specific Opa1 deletion
design: Bulk RNA-Sequencing of brain hemispheres of mice with colitis induced by intestinal epithelial-specific Opa1 deletion
library_construction_protocol: On day 24 after first tamoxifen administration, mice were sacrificed, followed by transcardial perfusion and isolation of the right brain hemisphere 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. VillinCreERT negative littermates treated with tamoxifen served as controls. 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.