|
| Status |
Public on Jan 04, 2020 |
| Title |
IL4c-2_R1 |
| Sample type |
SRA |
| |
|
| Source name |
Peritoneal macrophages
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
macrophage phenotype: Tissue resident, F4/80hi CD206-
|
| Treatment protocol |
Intra-peritoneal IL-4 complexes (i.e. 5μg of IL-4 and 25μg of anti-IL4 in 100ul sterile PBS) on Day 0 and Day 2.
All mice were treated with group-specific treatment as detailed in the Samples Section above and peritoneal cells were harvested on Day 4.
|
| Growth protocol |
C57BL/6 mice used in the experiments to identify the effects of IL-4 stimulation on accessible chromatin landscape were bred at the specific-pathogen free animal facility in NYU. C57BL/6 and BALB/c mice used in the experiment comparing strain effects on accessible chromatin landscape were purchased directly from Jackson laboratory and used immediately for experiments.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATACSeq was performed as described in Buenrostro et al (Nature Methods 2013). FACS-purified cells were spinned down at 1500rpm for 5 min at 4 degree Celsius and washed once with 50ul cold PBS. Cells were then lysed with 50ul lysis buffer (10 mM Tris-HCl, pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and the nuclei were immediately spinned down at 1500rpm for 10 min at 4 degree Celsius. The isolated cell nuclei were then incubated with 50ul of transposase reaction (25ul TD buffer, 2.5ul Tn5 enzyme, 22.5ul nuclease-free water)for 30 mins at 37 degree Celcius. Transposed DNA was purified using the QIAgen MinElute PCR Purification Kit following manufacturer's guide. TD buffer and Tn5 enzyme are from the Nextera DNA Sample Preparation kit (Illumina Cat #FC-121-1030).
Tranposed DNA was amplified using a low-cycle number PCR protocol and custom primers designed for multiplexing based on Buenrostro et al (Nature Methods 2013). Each sample underwent 14-16 cycles of PCR amplification. Amplified libraries of transposed DNA were purified with the QIAgen MinElute Kit and sequenced on the HiSeq 2000.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
genomic DNA (transposase-accessible)
Effect of IL-4 stimulation, Run1
ATACSeqIL4cMacs_union_peaks_max100.txt
M1_merged_runs_nochrMY_dedup.tdf
|
| Data processing |
Raw reads aligned to the reference mouse genomes mm9 (for experiments comparing IL-4 stimulated macrophages to unstimulated macrophages) and mm10 (for experiment comparing AAMs from C57BL/6 and BALB/c mice) using bowtie2 (v2.2.9), with the parameters --maxin 2000 and --local, while keeping all other parameters at default settings.
Reads with mappability score (MAPQ) <30 and duplicate reads, as well as reads mapping to mitochondrial DNA and chromosome Y, were removed.
Filtered reads were merged across all replicates from the same group. Peak calling was done on the merged reads for each group using PeaKDeck v1.1 (McCarthy and O'Callaghan, Bioinformatics 2014), using the parameters: central bin (-bin) 75bp, background bin (-back) 10000bp and step size (-STEP) of 25. Background probability distribution was generated using 100000 randomly selected sites (-npBack). Significance was defined using a p-value of less than 0.0001 (for experiments comparing IL-4 stimulated macrophages to unstimulated macrophages) and 0.00001 (for experiment comparing AAMs from C57BL/6 and BALB/c mice).
Genome_build: mm9, mm10
Supplementary_files_format_and_content: .txt files are count matrix, where each row is a unique accessible region; columns 1-4 correspond to the region genomic locations and subsequent columns correspond to a unique sample.
Supplementary_files_format_and_content: .tdf files are merged reads across biological replicates and converted to a Tiled Data Format (tdf) using IGVtools for visualization on the Integrative Genomics Viewer (IGV)
|
| |
|
| Submission date |
Jun 21, 2018 |
| Last update date |
Jan 04, 2020 |
| Contact name |
Mei San Tang |
| Organization name |
Washington University in St Louis
|
| Department |
Pathology & Immunology
|
| Street address |
660 S Euclid, Campus Box 8118
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63108 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE116107 |
Accessible chromatin profiles of IL-4 stimulated mouse peritoneal macrophages derived from different cellular lineages |
| GSE116108 |
Profiles of IL-4 stimulated mouse peritoneal macrophages |
|
| Relations |
| BioSample |
SAMN09464618 |
| SRA |
SRX4277560 |
