Single-cell RNA sequencing of murine lung in steady state, after IL-33 treatment or HDM treatment
Exp3: Mice were sensitized with 3 μg house dust mite extract (HDM - ALK) intratracheally and challenged two weeks later with PBS or 10 μg of HDM extract intranasally on 4 consecutive days. Lungs, bone marrow and blood was collected 24 hours after the last treatment. 3 mice per condition were pooled. Single cell suspensions of lung, bone marrow and blood were stained with FACS sort antibodies combined with CITEseq antibodies (TotalSeq-A)  in 25 μl mix containing PBS, 0.04% BSA,  TruStain FcX Block for 30 minutes on ice. The CITE-Seq panel contains 170 unique oligoconjugated antibodies and isotype controls. Different organs were tagged and pooled using TotalSeq-A hashtag antibodies. Sorting was performed to select single alive Ter119- CD3e- CD19- cells into a tube containing anti-PE CITEseq antibody to label CD101-PE+ cells. Per condition, first 45.000 lung cells were sorted, then 20.000 blood cells and 10.000 bone marrow cells into the same 1,5ml Eppendorf.  The DNA libraries were prepared using the GemCode Single Cell 3′ Gel Bead and Library kit, version NextGEM 3.1 (10× Genomics) according to the manufacturer's instructions with the addition of amplification primers (3 nM, 5′CCTTGGCACCCGAGAATT*C*C and 5′GTGACTGGAGTTCAGACGTGTGC*T*C) during cDNA amplification to enrich the TotalSeq-A cell surface protein and/or hashtag oligos. Size selection with SPRIselect Reagent Kit (Beckman Coulter, B23318) was used to separate amplified cDNA molecules for 3′ gene expression and cell surface protein construction. TotalSeq-A protein library construction including sample index Polymerase Chain Reaction (PCR) using Illumina's Truseq Small RNA primer sets and SPRIselect size selection was performed according to the manufacturer's instructions. 