GSM8778574: WT, FFS, biol repl 4; Mus musculus; RNA-Seq
Lungs were perfused priior to extraction and dissociated using collagenase media with Dnase for 1 hour followed by filtering in a 70uM filter to get a single cell suspension. Cells were rinsed with PBS and strained in a 40uM filter.  Dead cell removal was performed via negative selection using the EasySep Mouse Dead Cell Removal (Annexin V) kit. The flow through was centrifuged and resuspended in PBS (10% FBS, 1mM EDTA) and CD8+ T cells were isolated using the CD8+ T cell Isolation Kit as directed. RNA was extracted from isolated lung CD8+ T cells using TRI reagent (Zymo Research, Irvine, CA). Samples were digested on ice for 5 minutes with mechanical agitation of samples occurring 2-3 times. RNA was further extracted from samples using the Direct-zol Mini Prep kit as directed. RNA was shipped to MedGenome for sequencing via Takara SMART-Seq mRNA kit (100 read length).