|
| Status |
Public on Dec 17, 2015 |
| Title |
hMDM_GR_input |
| Sample type |
SRA |
| |
|
| Source name |
human monoycte derived macrophages
|
| Organism |
Homo sapiens |
| Characteristics |
pooled: yes
fully anonymized: yes
cell type: human monoycte derived macrophages
antibody: input
|
| Treatment protocol |
They were treated with either 100nM dexamethasone (Sigma) or ethanol vehicle for 2h.
|
| Growth protocol |
CD14+ monocytes were extracted from whole blood donated by healthy volunteers using MACS separation (Miltenyi). They were differentiated in culture for 1 week in the presence of recombinant human CSF1 (10000U/ml),penicillin/streptomycin, Glutamax and 10% fetal calf serum then replated in fresh medium, also supplemented as above, for the time series experiment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
rinsed with PBS, fixed in 1% formaldehyde for 7.5min at RT, lysed and sonicated to fragment size 400-600bp. GR-DNA complexes were then isolated from soluble chromatin using Sigma Imprint anti GR monoclonal antibody , crosslinks reversed at 65 degrees and DNA cleaned up using Minielute columns(Qiagen)
ChIP DNA samples were split into 3 aliquots and blunt ended with Klenow (Roche), PNK (NEB) and T4 DNA polymerase (Roche). An overhanging A base was added using Klenow (-exo) (NEB) and Illumina adapters ligated overnight at 16 degrees C with T4 DNA ligase (NEB). The IP samples were recombined after ligation and then split again into 7 aliquots. Libraries were amplified from each of these aliquots using Illumina Tru-seq multiplex primers and Phusion high-fidelity DNA polymerase (NEB) and the resulting material pooled and sequenced by Edinburgh Genomics on a Hiseq-2500.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ChIP DNA
|
| Data processing |
Base calls were generated using CASAVA 1.8
Trimming of adapters, Trimmomatic v0.27, ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Alignment, Bowtie2 for PE sequencing (defaults)
Formation of tag directories, HOMER
peak calling and removal of low absolute value peaks(cmd = ./homer/bin/findPeaks ./homer/homer_out/combined_1119_dexAb_tags/ -style factor -o auto -i ./homer/homer_out/combined_dex_input_1119_tags/), production of bedGraphs, HOMER.
Formation of bedGraph for visualization, HOMER
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph for visualization of treated IP data, tab-delimited peak file
|
| |
|
| Submission date |
Sep 29, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Alasdair W Jubb |
| E-mail(s) |
alasdair.jubb@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Roslin Institute
|
| Lab |
Hume lab / Bickmore lab (MRC HGU)
|
| Street address |
The Roslin Institute
|
| City |
Easter Bush |
| State/province |
Midlothian |
| ZIP/Postal code |
EH25 9RG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE61878 |
Genome wide binding of glucocorticoid receptor in dexamethasone treated human monocyte derived macrophages |
| GSE61881 |
Divergent transcriptional activation by glucocorticoids in mouse and human macrophages is the result of gain and loss of enhancers |
|
| Relations |
| BioSample |
SAMN03084137 |
| SRA |
SRX716945 |
