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| Status |
Public on Oct 18, 2021 |
| Title |
ILC2_Id1-Tg2_HDM |
| Sample type |
SRA |
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| Source name |
Lung ILC2
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| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
genotype/variation: Id1tg/WT
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| Treatment protocol |
Mice were administered 80 mg of HDM (Greer) in 40 mL PBS or PBS alone intranasally (IN), every 7 days for 3 weeks. Tissues were collected 3 days after the final treatment.
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| Growth protocol |
Id1tg/tg and Id1tg/WT mice were as described (PMID: 10567549). For Id1tg/WT mice, Id1tg/tg mice were crossed with C57BL/6 WT mice (Jackson Laboratories, Bar Harbor, ME) and bred in-house. Co-housed experimental controls were Id1WT/WT male or female littermates, in specific pathogen-free conditions at Cornell University or the University of Washington, in accordance with protocols approved by the respective Institutional Animal Care and Use Committees.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
ATAC raw data was mapped to the Mus musculus 10 (mm10) genome using the Burrows-Wheeler Alignment (BWA)-mem algorithm (PMID: 19451168) and peaks were called using MACS2 (PMID: 18798982).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files were generated based on MACS2 pileup bedgraphs
Supplementary_files_format_and_content: narrowPeak files were generated by MACS2
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| Submission date |
Oct 14, 2021 |
| Last update date |
Oct 18, 2021 |
| Contact name |
Elia D. Tait Wojno |
| E-mail(s) |
etwojno@uw.edu
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| Organization name |
University of Washington
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| Department |
Department of Immunology
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| Street address |
750 Republican St.
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL30172 |
| Series (1) |
| GSE185950 |
E-protein inhibition in ILC2 development shapes the function of mature ILC2s during allergic airway inflammation |
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| Relations |
| BioSample |
SAMN22310694 |
| SRA |
SRX12626598 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5627736_ILC2_Id1-Tg2_HDM.bw |
120.2 Mb |
(ftp)(http) |
BW |
| GSM5627736_ILC2_Id1-Tg2_HDM_peaks.narrowPeak.gz |
772.7 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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