NextSeq 550 paired end sequencing; RNA-seq data of lung resident T-cells from mice following house dust mite (HDM) sensitisation and subsequent influenza virus challenge
RNA-seq data of lung resident T-cells from mice following house dust mite (HDM) sensitisation and subsequent influenza virus challenge
Single cell suspension was prepared from lungs using the mouse lung dissociation kit (Miltenyi Biotec) and passed through a 70m strainer. Cells were labelled with fluorochrome conjugated antibodies and different cell populations were sorted in RLTplus buffer (Qiagen) using BD FACS ARIAIII and FACS DIVA software (BD Biosciences). For sorting of tetramer (NP) positive cells, single cell suspensions from lungs were enriched for CD8+ T cells using EasySep Mouse CD8+ T Cell Isolation Kit (Stem Cell technologies) prior to staining with antibodies. 6-8 weeks old Balb/cJRj mice (Janvier Labs) were subjected to  intranasal (i.n.) sensitization/challenge regimen over a span of three weeks using house dust mite (HDM) extract from Dermatophagoides pteronyssinus (Stallergenes Greer, USA). In the first and third week, the mice were sensitized/challenged with 25mg of HDM extract in 20ml of phosphate-buffered saline (PBS) daily for 5 consecutive days. 21 days after the last HDM dose, the mice were immunized once with a sublethal dose of X47 H3N2 flu virus (0.1LD50, 1 PFU in 30ml of PBS) or PBS only as control via the i.n. route. Control groups of mice that were only sensitized/challenged with HDM, only immunized with X47 H3N2 flu virus or PBS-treated controls were also included.  Seven weeks after the immunization with X47 (day 87), mice were sacrificed to study influenza- and HDM-specific memory immune responses. Total RNA was extracted from sorted T cell population using RNeasy plus micro kit (Qiagen) following manufacturers instructions Libraries were generated using QIAseq FX single cell RNA library kit (Qiagen) according to manufacturers instructions. Libraries were validated on the Fragment Analyzer platform (AATI) and concentrations were determined using the Quant-iT dsDNA HS assay kit on the Qubit fluorometer (Thermo Fisher Scientific) as well as with qPCR using QIAseq Library Quant Assay kit.