|
| Status |
Public on Feb 28, 2016 |
| Title |
ChIP-Seq of PU1 in TPP Macrophages |
| Sample type |
SRA |
| |
|
| Source name |
primary human macrophages
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: peripheral blood derived macrophage
treatment: GM-CSF for 72h and additional 72h with GM-CSF + TNF-a + PGE2 + Pam3CSK4
chip antibody: PU.1 polyclonal rabbit antibody (SantaCruz, sc-352)
|
| Treatment protocol |
Fractions of baseline macrophages were further stimulated for 72h in culture medium with the addition of three stimulatory cocktails containing IFN-γ (200U/ml), IL-4 (500U/ml) or a combination of TNF-α (800U/ml), PGE2 (1µg/ml) and Pam3CSK4 (1µg/ml) to differentiate macrophages into subtypes
|
| Growth protocol |
MACS beads isolated CD14 primary human monocytes were cultured for 72 h with granulocyte/macrophage colony stimulating factor (GM-CSF; 500U/ml) in RPMI1640 medium containing 10 % fetal calf serum (FCS) and 1% Penicillin/Streptomycin to generate unstimulated baseline macrophages
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin from 0.5x10E6 macrophages was cross-linked for ChIP reactions with 1 % formaldehyde. Nuclei were lysed and chromatin was sheared with the Covaris S220 ultrasound system. For ChIP antibody reactions a PU.1 polyclonal rabbit antibody was used (SantaCruz, sc-352). Washing and DNA purifications as well as size selections were performed with AMPure XP SPRI beads (Beckman Coulter).
Published library construction protocol was adapted (Blecher-Gonen et al., Nature Protocols, 2013) and performed with 0.5 ng ChIP DNA. Barcodes from the NEXTflex adapter oligonucleotides kit (Bioo Scientific) were ligated to the ChIP-seq DNA fragments. Final DNA purification with additional size selection was performed with SPRI beads. ChIP-seq libraries were sequenced on the HiScanSQ/Hiseq 1000 sequencer (Illumina)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
Basecalls performed using CASAVA software version 1.8
ChIP-seq reads were aligned to the NCBI Build GRCh36/UCSC Build hg18 genome assembly using Bowtie v0.12.8 with following options: -t -q -e 70 -l 28 -n 2 --best --maxbts 125 -S
After ChIP-seq quality control using HOMER v4.3, reads of biological replicates were pooled, and peaks were called using HOMER 4.3 with findPeaks.pl script with pooled Input samples used as background control files using default options and following additional settings: -style factor
Annotation of peak positions and counting of normalized tag counts in called regions was performed with HOMER v4.3 annotatePeaks.pl script.
Genome_build: hg18
Supplementary_files_format_and_content: Annotated HOMER called peak positions in tab-delimited text format
|
| |
|
| Submission date |
May 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Joachim Schultze |
| E-mail(s) |
j.schultze@uni-bonn.de
|
| Organization name |
LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)
|
| Department |
Genomics and Immunoregulation
|
| Street address |
Carl-Troll-Strasse 31
|
| City |
Bonn |
| State/province |
NRW |
| ZIP/Postal code |
53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18460 |
| Series (2) |
| GSE66594 |
Epigenomic landscapes of human inflammation associated macrophages [ChIP-seq] |
| GSE66595 |
Epigenomic landscapes of human inflammation associated macrophages |
|
| Relations |
| BioSample |
SAMN03653333 |
| SRA |
SRX1023793 |
