Bulk RNA-Sequencing of brain hemispheres of mice with T-cell transfer colitis
Bulk RNA-Sequencing of brain hemispheres of mice with T-cell transfer colitis
Three weeks after T cell transfer, mice were sacrificed, transcardially perfused with PBS and right brain hemispheres collected Naïve CD4+ T cells were isolated from the spleen of C57BL/6J mice using magnetic-based cell separation and subsequently, 1 million CD4+ CD25- cells were injected intraperitoneally into Rag1-/- mice (on C57BL/6J background). Rag1-/- mice injected with 200µL PBS served as controls. 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.