Tifa-/- vs Tifa+/+ small intestinal organoids stimulated with water, IL-22, IL-22+ADP-Heptose
Small intestinal tissue was obtained and prepared for organoid generation according to established protocols. After removing the medium, organoids were washed 3 x with 1ml PBS w/o Ca++/Mg++ and then collected for RNA extraction. Total RNA was isolated using the PeqGold microspin total RNA Kit following manufacturers' instructions (Peqlab/VWR). 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.