scRNA-seq analysis of enriched CD4+ T cells from lung tissues after single or combined AIT treatment strategies with AhR agonist 10-Cl-BBQ in a murine model of OVA-induced allergic airway inflammation (AAI)
scRNA-seq analysis of enriched CD4+ T cells from lung tissues after single or combined AIT treatment strategies with AhR agonist 10-Cl-BBQ in a murine model of OVA-induced allergic airway inflammation (AAI)
To isolate pulmonary leukocytes, lungs were removed and chopped into small pieces. Digestion of lung tissue was performed in RPMI medium (Thermo Fisher Scientific) containing 1 mg/mL collagenase A and 100 µg/mL DNAse I (both from Sigma-Aldrich, Merck, Darmstadt, Germany) for 30 min at 37° C. Digested lung tissue was mashed trough a 70 µm cell strainer. Cells were pelleted (400 g, 4 °C, 5 min), resuspended in 5 mL 40% percoll in RPMI (v/v) solution, underlayered with 5 mL 80% percoll solution, and centrifuged (1600 g, RT, 15 min). Lymphocytes were collected from the interphase and washed with PBS. CD4+ T cells were enriched from pulmonary lymphocytes with the EasySepTM Mouse CD4+ T cell Isolation Kit and the EasyEightsTM EasySepTM Magnet (both from STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's instructions.  All libraries for scRNA-seq were prepared according to the manual (CG000315 Rev A) of the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Dual Index) provided by 10x Genomics B.V. (Leiden, The Netherlands). A cell recovery of 10.000 was targeted. All libraries for scRNA-seq were prepared according to the manual (CG000315 Rev A) of the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Dual Index) provided by 10x Genomics B.V. (Leiden, The Netherlands). A cell recovery of 10.000 was targeted.