Raver2 and Setd2 knockdown of mouse mTECs in a 3D orgonatypic co-culture system

Aire-sensitive genes escape the inclusion of alternative splicing events that Raver2 and Setd2 promote in mTECs Two shRNAs against Raver2 and Setd2 were cloned into the pLKO.3G vector containing an eGFP cassette (Addgene #14748). Ultra-high titer lentiviruses were produced into HEK293T cells by using the calcium phosphate co-transfection method for each specific subcloned lentiviral pLKO.3G. Primary mouse mTECs (CD45- EpCAM+ Ly51- I-A/E+) were seeded at the top of the 3D organotypic co-culture setup that we prepared in seeding human dermal fibroblasts into a fibrin gel made of fibrinogen, thrombin and aprotinin. The infection was directly performed with the addition of the concentrated shRNA-bearing lentiviruses that we produced against Raver2, Setd2 or the Ctr LacZ. Five days after infection, the fibrin gel was processed to evaluate the impact of Raver2 and Setd2 knockdown on the inclusion of alternative splicing events of Aire-sensitive and neutral genes by RNA-seq. We sorted by flow cytometry DAPI- I-A/E+ GFP+ cells corresponding to viable lentivirus-infected mTECs.