Single-cell RNA sequencing of murine lung in steady state, after IL-33 treatment or HDM treatment
Single-cell RNA sequencing of murine lung in steady state, after IL-33 treatment or HDM treatment
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.  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. 