GSM5823429: FRT CD4 Tbet-/- rep4; Mus musculus; RNA-Seq
Five samples for each group were prepared by pooling three mice per sample to accumulate enough cells for RNA isolation. Lymphocytes were isolated from the FRT of mice 17 days post infection i.vag. with Chlamydia muridarum via collagenase IV digestion and a percoll gradient. Cells were then stained for flow sorting using Zombie Yellow (BioLegend) and antibodies for surface markers APC-B220 (RA3-6B2, eBioscience), APC-F4/80 (BM8.1, Tonbo Biosciences), APC-CD11b (M1/70, Tonbo Biosciences), APC-CD11c (N418, Tonbo Biosciences, eF450-CD4 (RM4-5, eBioscience), and PerCP-Cy5.5-CD8a (53-6.7, eBioscience). CD4 T cells were collected passing through single cell, live, dump (B220, F4/80, CD11b, CD11c) negative, and CD4+CD8- gates. RNA was isolated using the Qiagen RNeasy Mini Kit. Barcoded sequencing libraries were prepared using the 3' mRNA-seq QuantSeq FWD kit (Lexogen, Vienna, Austria). Library preparation was performed by the UC Davis DNA Tech Core.