GSM5627731: Th2_HDM; Mus musculus; ATAC-seq
For flow sorting, the entire lung was collected for tissue digestion. Lungs were digested with 20 µg/mL DNAase (Roche) and 2 mg/mL collagenase D (Roche) or 1 U/mL Liberase TL (Roche) at 37°C. Single cell suspensions were mashed through a 40 μm strainer. Lungs were RBC lysed with ACK lysis buffer (Lonza). For ILC2s and CD4+ T cells, single cell suspensions were sorted using a 4-laser FACS Fusion (BD Biosciences) (WT ILC2 and CD4+ T cell ATAC-seq), or a 4-laser and 3-laser Aria (BD Biosciences) (Id1WT/WT versus Id1tg/WT ILC2 ATAC-seq ) with an 85 μm nozzle. All leukocyte populations were gated as singlet, live (Live/Dead Aqua-) CD45+ leukocytes (by FSC-A/SSC-A), ILC2s were gated as lineage (lin) (CD3/CD5orTCRβ/CD11b/CD11c/CD19/NK1.1/TCRγδ)-CD90.2+/-CD127+Klrg1+/-ST2+/-, and CD4+ T cells were gated as TCRβ+, lineage (lin) (CD11b/CD11c/CD19/NK1.1/TCRγδ)-CD4+, further subdivided as Foxp3+ Tregs, Foxp3- CD44hiCD62Llo activated, and Foxp3-Gata3+, or ST2+ Th2 cells. Following flow sorting, ILC2s or CD4+ T cells were resuspended in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40 Lysis Buffer (IGEPAL) in H2O) with 1x protease and 2 U/mL SuperaseIN RNase (ThermoFisher) inhibitors. Permeabilized cells were resuspended in storage buffer (50 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 40% glycerol, 0.1 mM EDTA, pH 8.0, plus 40 U RNase inhibitor in H2O) and stored at -80 °C. Permeabilized cells were washed in PBS, then incubated in Transposition Mix (Nextera DNA Sample Preparation kit, Illumina), as previously described (PMID: 25559105). Following transposition, WT CD4+ T cells and ILC2 samples from PBS- or HDM-treated mice were purified using the Monarch Gel Extraction kit (NEB). Libraries were amplified using Nextera PCR oligos (Illumina) and Next High-Fidelity PCR Master Mix (NEB) for 12-14 cycles, cleaned up using the Monarch Gel Extraction kit and AMPure beads (Beckman Coulter), and sequenced with 2x 41 nucleotide paired-end reads on the NextSeq500 (Illumina). For Id1WT/WT and Id1tg/WT ILC2 analysis from HDM-treated mice, following transposition, samples were purified using the Qiagen MinElute PCR Purification kit. All libraries were amplified using PCR oligos as described previously (PMID: 24097267) and NEB Next High-Fidelity PCR Master Mix (NEB) for 11 cycles. Libraries were purified using the Qiagen MinElute PCR Purification kit and treated with the Free Adapter Blocking Reagent (Illumina) with 1.8x Agencourt AMPure XP bead size selection (Beckman Coulter).  Sequencing was performed with a target of 50M reads per library using 59 base paired-end reads on a P2 flowcell on the NextSeq2000 (Illumina).