Bulk RNA-Sequencing of brain hemispheres of mice with anti-CD3-induced small intestinal inflammation
Bulk RNA-Sequencing of brain hemispheres of mice with anti-CD3-induced small intestinal inflammation
Mice were sacrificed after 6 or 24 hours of aCD3 or 24 hours of isotype control, respectively. After transcardial perfusion, right brain hemispheres were dissected and isolated RNA sent for sequencing Male C57BL/6J mice were injected intraperitoneally with 30µg purified NA/LE Hamster anti-mouse CD3e antibody (BD Bioscience) in PBS, whereas control mice received 30µg Hamster IgG NA/LE isotype control (BD Bioscience) RNA isolation was performed using the RNeasy Mini kit (QIAGEN) following the manufacturer'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.