GSM7099899: GB7041 (WT Veh 1); Mus musculus; RNA-Seq
Single cell suspension were prepared from the brain and spinal cords of mixed congenic receipient EAE mice via digestion with 1 U/ml Liberase TM (Roche) and 0.3 U/ml DNase I (Roche) in incomplete RPMI1640 media for 30 minutes at 37C. The tissues were mechanically disrupted via repetitive pipetting and filtered through a 70μm cell strainer (Fisher Scientific). The resulting cell suspension was centrifuged through a percoll density gradient (38% - 70%) and mononuclear cells were collected from the interphase. CNS-infiltrating CD4 T effector cells (i.e., CD45.1+CD45.2+ CD3+CD4+CD44 high wild type T cells or CD45.2+CD3+CD4+CD44 high Cre-ERT2 Gata3 Fl/Fl T cells) were isolated by FACS sorting. RNA libraries were prepared for sequencing using standard Illumina protocols