RNA-seq of T cells form house dust mite mouse model of bronchoalveolar lavage (BAL), medLN and lung tissues
RNA-seq of T cells form house dust mite mouse model of bronchoalveolar lavage (BAL), medLN and lung tissues
Wild-type C57BL/6J mice (6–8 weeks old) were sensitised intranasally under light isoflurane anaesthesia with 1 µg house dust mite (HDM) extract in 40 µL PBS, followed seven days later by intranasal challenges of 10 µg HDM once daily for five consecutive days. Four days after the final challenge (day 15), mice were euthanised and BAL was collected by two sequential flushes of the airways with 1 mL PBS and , medLN, lung tissue were collected at the same time. Cells were stained for surface markers and labelled with TotalSeq-C hashtag antibodies (BioLegend) for sample identification. CD3⁺CD4⁺ T cells were sorted into FCS on a BD FACSAria Fusion, washed, and resuspended in cold PBS for single-cell capture. No bulk nucleic acid extraction was performed.  Sorted CD3⁺CD4⁺ T cells in cold PBS were loaded onto the 10x Genomics Chromium controller for droplet partitioning with barcoded gel beads. Cell capture was performed by the National Genomics Infrastructure (NGI), SciLifeLab, Stockholm. The full-length cDNA was used to generate three libraries per reaction: (i) a 5′ gene-expression library, (ii) a paired T-cell receptor (TCR) V(D)J libraryand (iii) a cell-hashing library used for sample demultiplexing. Libraries were prepared with the 10x Genomics Chromium Single Cell 5′ v1 Immune Profiling reagent kit. Library preparation was performed by NGI, SciLifeLab, Stockholm. 