|
| Status |
Public on Feb 13, 2014 |
| Title |
PU.1_M1_Macro_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: monocyte derived macrophages
initial differentiation: GM-CSF
activation stimuli: IFNg, 72h
chip antibody: anti PU.1 (Santa Cruz Biotechnology, sc-352X, lot D2011)
|
| Treatment protocol |
To further polarize macrophages into different subtypes the following stimuli were used: M1 (3days 500IU/ml rhGM-CSF + 200IU/ml rhIFNy); M2 (3days 500IU/ml rhGM-CSF + 1000IU/ml rhIL-4); TPP (3days 500IU/ml rhGM-CSF + Pam3CSK4 (P3C, 1 µg/ml) + prostaglandine E2 (PGE2, 1 µg/ml) + TNFa (800 IU/ml))
|
| Growth protocol |
For differentiation primary human monocytes into M0 macrophages, cells were cultivated for 3 days in RPMI1640 medium + 10%FCS + Pen/Strep + 500IU/ml rhGM-CSF
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed or unfixed chromatin out of primary human macrophage lysates was sheared and histone-DNA or TF-DNA complexes isolated with antibody.
Multiplex DNA libraries of both H3K4me3 as well as PU.1 bound DNA were generated using Illumina’s ChIP-Seq Sample Preparation Kit (Illumina; # IP-102-1001) and the Multiplexing Sample Preparation Oligonucleotide Kit (Illumina; # PE-400-1001) using approximately 10 ng DNA following the manufacturer’s instructions. Briefly, purified DNA ends were repaired using PNK and Klenow enzyme, followed by treatment with Klenow exo minus polymerase to generate a protruding 3′ A base used for adaptor ligation. Next, size selection of libraries was performed as follows: DNA libraries were agarose gel purified, DNA fragments with approximately 220 bp size excised and eluted using QIAquick Gel Extraction kit (Qiagen). After subsequent adapter ligation to the repaired ends, an amplification step was performed for 5 cycles with PCR primers 1.1 and 2.1 (Illumina; # IP-102-1001). During a second 13 cycles amplification step multiplex PCR primers were added to the DNA libraries to construct multiplex sequencing libraries. For PU.1 DNA libraries multiplex PCR primers were added directly after adapter ligation to the amplification mix and 18 cycles of amplification were performed. After amplification steps DNA was purified.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiScanSQ |
| |
|
| Description |
raw file: D118TACXX_human_CGATGT_M1_PU1_SR_001.fastq.gz
|
| Data processing |
Basecalls performed using CASAVA software version 1.7 (for histone datasets) and 1.8 (for PU.1 datasets)
ChIP-seq reads were aligned to the NCBI Build 36 (hg18) genome assembly using ELAND (Illumina Pipeline) at its default setting and were exported in .bam file format
Genome_build: NCBI36
|
| |
|
| Submission date |
May 22, 2013 |
| 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 |
GPL15456 |
| Series (2) |
| GSE47188 |
Transcriptome-based network analysis reveals a spectrum model of human macrophage activation [ChIP-Seq] |
| GSE47189 |
Transcriptome-based network analysis reveals a spectrum model of human macrophage activation |
|
| Relations |
| BioSample |
SAMN02169219 |
| SRA |
SRX286018 |
