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| Status |
Public on Jun 25, 2019 |
| Title |
RNA_ILC2_NMU_4h_rep2 |
| Sample type |
SRA |
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| Source name |
mouse primary lung ILC2s
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
Sex: female
age: 6-12 weeks
culture condition: 4 hour in vitro culture (growth protocol 5)
genotype: WT
lab data id: t05
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| Growth protocol |
growth protocol 1: FastATACseq in vitro: Lin- CD3- TCRβ- Thy1+ CD127+ KLRG1+ ILC2s were purified from lungs of mice by cell sorting (>98% purity; FACSAria III or FACSAria Fusion; Becton Dickinson) and cultured 2-4 weeks in the presence of recombinant mouse IL-7 (10 ng/ml; R&D) and recombinant human IL-2 (100 U/ml). Then, IL-2 and IL-7 were removed from ILC2 culture and additionally cultured for 4 hr before in vitro stimulation.1 x 10^4 cells were cultured with various stimulants including IL-33 (10 ng/ml), NMU (10 ng/ml), CGRP (10 ng/ml) for 4hr. These were then collected and processed for subsequent ATAC-seq. Cells were maintained in supplemented tissue culture medium (RPMI-1640 with 10% fetal calf serum, 50µM β-Mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin; Life Technologies) and cultured at a density of 5 x 10^4 cells/ml in U bottomed, 96 well tissue culture-treated plates (CoStar).
growth protocol 2: scRNAseq ex vivo: 1.3 x 10^4 Lin- CD3- TCRβ- Thy1+ ILCs or CD3+ TCRb+ CD4+ Th cells were purified from lungs of mice by cell sorting (>98% purity; FACSAria III or FACSAria Fusion; Becton Dickinson) and immediately pocessed for subsequent scRNA-seq (10X Genomics).
growth protocol 3: mRNAseq ex vivo: 2-5 x 10^4 CD3+ TCRβ+ CD4+ CD62L+ CD44low CD25- naive Th cells were purified from mediastinal lymph nodes (mLNs) of mice by cell sorting (>98% purity; FACSAria III or FACSAria Fusion; Becton Dickinson) and stored in Trizol reagent (Invitrogen) for subsequent RNA isolation.
growth protocol 4: mRNAseq ex vivo: 2-5 x 10^4 Lin- CD3- CD4- TCRβ- Thy1+ CD127+ KLRG1+ ILC2s or CD3+ TCRb+ CD4+ ST2+ Th2 cells were purified from lungs of mice by cell sorting (>98% purity; FACSAria III or FACSAria Fusion; Becton Dickinson) and stored in Trizol reagent (Invitrogen) for subsequent RNA isolation.
growth protocol 5: mRNAseq in vitro: Lin- CD3- TCRβ- Thy1+ CD127+ KLRG1+ ILC2s were purified from lungs of mice by cell sorting (>98% purity; FACSAria III or FACSAria Fusion; Becton Dickinson) and cultured 2-4 weeks in the presence of recombinant mouse IL-7 (10 ng/ml; R&D) and recombinant human IL-2 (100 U/ml). Then, IL-2 and IL-7 were removed from ILC2 culture and additionally cultured for 4 hr before in vitro stimulation.3 x 10^4 cells were cultured with various stimulants or inhibitor, including IL-33 (10 ng/ml), NMU (10 ng/ml), CGRP (10 ng/ml), Dibutyryl-cAMP (100 μM), and Gaq inhibitor (10 μM) for 4hr. These were then collected and resuspended in Trizol ragent (Invitrogen) for subsequent RNA isolation. Cells were maintained in supplemented tissue culture medium (RPMI-1640 with 10% fetal calf serum, 50µM β-Mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin; Life Technologies) and cultured at a density of 1.5 x 10^5 cells/ml in U bottomed, 96 well tissue culture-treated plates (CoStar).
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| Extracted molecule |
total RNA |
| Extraction protocol |
FastATAC-seq: 1 X 10^4 cells were pelleted and washed with 50 μl 1× PBS. After pelleting the nuclei by centrifuging at 500 × g for 10 min, the pellets were re-suspended in 50 μl transposase mixture (25 µl of 2x TD buffer, 2.5 µl of TDE1, 0.5 µl of 1% digitonin, 22 µl of nuclease-free water) (#FC-121-1030, Illumina; #G9441, Promega). The reaction was incubated at 37°C with shaking at 300 rpm for 30 min. The fragmentalized DNAs were then purified using a QIAGEN MinElute kit and amplified with 10 or 11 cycles of PCR based on the amplification curve, and the paired-end libraries were purified using a QIAGEN PCR cleanup kit
scRNA-seq: Freshly sorted ILCs and Th cells (13,000 cells each) were encapsulated into droplets, and libraries were prepared using Chromium Single Cell 3’ Reagent Kits v2 according to manufacturer’s protocol (10X Genomics).
RNA-seq: Total RNA was extracted from 2-5 x 10^4 cells by Direct-zol™ RNA MicroPrep (Zymo Research). Single-end libraries were prepared from 0.1-0.5 μg of total RNA using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530S)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
171206_171213_combined
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| Data processing |
ATAC-seq: Raw sequencing data were processed with CASAVA 1.8.2 to generate FastQ files. Reads were mapped to the mouse genome (mm9 assembly) using Bowtie 0.12.8. In all cases, redundant reads were removed using FastUniq (Xu et al., 2012), and customized Python scripts were used to calculate the fragment length of each pair of uniquely mapped paired-end (PE) reads. Only one mapped read to each unique region of the genome that was less than 175 bp was kept and used in peak calling.
ATAC-seq: Regions of open chromatin were identified by MACS (version 1.4.2) using a p-value threshold of 1 × 10−5.
RNA-seq: Sequence reads from all samples were processed and aggregated using Cell Ranger.
RNA-seq: Raw sequencing data were processed with CASAVA 1.8.2 to generate FastQ files. Reads of 50 bases were mapped to mouse genome mm9 using TopHat 2.1.0.
Gene expression values (FPKM; fragments per kilobase exon per million mapped reads) were calculated with Cufflinks 2.2.1
Genome_build: ATAC-seq: mm9
Genome_build: RNA-seq: mm10
Supplementary_files_format_and_content: bw for ATAC-seq and mRNA-seq peak files
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| Submission date |
May 30, 2019 |
| Last update date |
Jun 25, 2019 |
| Contact name |
Yuka Kanno |
| E-mail(s) |
kannoy@mail.nih.gov
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| Phone |
301-402-3008
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| Organization name |
NIH
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| Department |
NIAMS
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| Lab |
LCBS-MIIB
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| Street address |
10 Center Drive, Rm13C120
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-1930 |
| Country |
USA |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE131996 |
Neuropeptide CGRP limits ILC2 responses and constrains type 2 inflammation |
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| Relations |
| BioSample |
SAMN11895876 |
| SRA |
SRX5936374 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3834140_RNA_ILC2_NMU_4h_rep2_H037.bw |
163.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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