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.  Exp2: Mice were treated with PBS, 1 x 100 ng IL-33 intratreacheally or 3 x 100 ng IL-33 intratracheally. Mice were sacrificed 24h after the last treatment and single cell lung suspensions were created. Cells were incubated with FACS antibodies as well as TotalSeq A hashtag antibodies. 12000 CD45-, 3000 CD45+ CD11c+ SiglecF+ alveolar macrophages, 3000 CD45+ lineage- CD11c- SiglecF- CD90+ ST2+ ILC2s and 12000 AM and ILC2 excluded CD45+ cells were FACS purified per mouse and pooled together per condition. Cells were diluted to a final concentration of 1000/μl and loaded on the 10X Chromium device.