GSM9598145: CD4Tcric_rep1; Mus musculus; RNA-Seq
To distinguish tissue-resident from circulating leukocytes, mice were intravenously injected with PE-conjugated anti-CD45 antibody and sacrificed three minutes later. Lungs were perfused with cold PBS via the right ventricle and mechanically dissociated in digestion buffer containing collagenase type III and DNase I. Samples were incubated at 37°C for 30 minutes with agitation. Mononuclear cells were isolated by Percoll density centrifugation and filtered through a 70-μm cell strainer. CD4⁺ T cells were subsequently sorted and subjected to single-cell RNA sequencing. Single-cell RNA sequencing libraries were prepared using the Chromium Single Cell 3′ Reagent Kits v3.1 (10X Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 platform using paired-end reads.