|
| Status |
Public on Sep 15, 2020 |
| Title |
WT IL-4 24h BMDM Egr2 ChIP-seq replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Bone marrow derived macrophages
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: Bone marrow
cell type: Bone marrow derived macrophages (BMDM)
genotype: WT
treatment: IL4
time: 24h
chip antibody: EGR2
|
| Treatment protocol |
ATAC-seq, ChIP-seq and gene expression measurements were performed in macrophages differentiated with M-CSF, on the 6th day cells were exposed to IL-4 (20ng/ul) for the indicated period of time.
|
| Growth protocol |
Isolated bone marrow-derived cells were differentiated for 6 days in the presence of L929 supernatant. Cells were either exposed to IL-4 (5ng/ml) during the whole differentiation process or polarized on the 6th day of the differentiation with IL-4 (20ng/ml) for the indicated period of time.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RNA-seq, wild type and Egr2fl/fl macrophages were differentiated in the presence of M-CSF using L929 cell supernatants for 6 days. On the 6th day cells were re-plated and treated with IL-4 (20ng/ml) for 24 hours or left untreated. Poly-A tailed RNA molecules were pulled down with poly-T oligo attached magnetic beads. Following purification, mRNA was fragmented then cDNA was generated by random primers and SuperScript II enzyme (Life Technologies). Second strand synthesis was performed followed by end repair, single ‘A’ base addition and ligation of barcode-indexed adaptors to the DNA fragments. ChIP was performed essentially as previously described (Daniel et al., 2014b), (Daniel et al., 2014a). ATAC-seq was carried out as described earlier with minor modification (Buenrostro et al., 2013).
For RNA-seq adapter-specific PCRs were performed to generate sequencing libraries. Approximately 2.5ug was used for library preparation with TruSeq RNA Sample Preparation Kit (Illumina). Libraries were size selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by QIAquick Gel Extraction Kit (Qiagen). Libraries were sequenced on HiSeq 2500 instrument.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
mm_BMDM_ChIPseq_WT_EGR2_IL4_24h_rep1
|
| Data processing |
RNA-seq: Reads were aligned to the mouse reference genome (mm10) using the default parameters of HISAT2. Rsubread’s featureCounts function was used to quantify the reference transcriptome (Gencode: M21) and generate raw count table (useMetaFeatures=TRUE and allowMultiOverlap=TRUE).
The primary analysis of ATAC-seq and ChIP-seq reads were carried out using ChIP-seq command line pipeline [Barta, 2011]. Briefly, reads were mapped to the mouse reference genome (mm10) using the default parameters of BWA MEM aligner. Low mapping quality reads (MAPQ<10), reads mapping to ENCODE mouse blacklisted [ref] regions and duplicated reads are discarded from downstream analysis, using samtools rmdup and bedtools intersectBed. Open chromatin regions or putative binding sites were determined using MACS2 at 5% false discovery rate (FDR). Coverage profiles were calculated using deeptools2 bamCoverage (--binSize 10 --smoothLength 40 --normalizeUsing RPGC –effectiveGenomeSize 2652783500).
Genome_build: mm10
Supplementary_files_format_and_content: RNA-seq: Raw and normalized (edgeR, cpm) count matrices. ChIP-seq, ATAC-seq: Normalized coverage files (deeptools2, bigwigs).
|
| |
|
| Submission date |
May 21, 2020 |
| Last update date |
Sep 16, 2020 |
| Contact name |
Laszlo Halasz |
| E-mail(s) |
laszlo.halasz@jhmi.edu
|
| Phone |
7276416811
|
| Organization name |
Johns Hopkins University
|
| Department |
Institute for Fundamental Biomedical Research
|
| Lab |
Nagy Lab
|
| Street address |
600 5th Ave S
|
| City |
Saint Petersburg |
| State/province |
Florida |
| ZIP/Postal code |
33701 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE151015 |
The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the stable epigenomic program of alternative macrophage polarization |
|
| Relations |
| BioSample |
SAMN14994259 |
| SRA |
SRX8379014 |
