|
| Status |
Public on Jan 19, 2018 |
| Title |
AcH4 Human Aortic Vascular Smooth Muscle Cells 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Human Aortic Vascular Smooth Muscle Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HuAoVSMC
antibody: pan-AcH4
|
| Growth protocol |
HuAoVSMC (ScienCell; https://www.sciencellonline.com/human-aortic-smooth-muscle-cells.html) were cultured according to provider's instructions using their recommended complete human smooth muscle cell medium. All biological replicates were used at passage 3-4.
See protocols in SAMPLES
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was conducted using the Chromatin Immunoprecipitation kit (Millipore, 17-295) according to the manufacturer's instructions. Briefly, 1x 106 cells were used per ChIP reaction. Cultured cells were fixed in 1% formaldehyde at 37 °C in 5% CO2 for 10 minutes by adding formaldehyde directly to the medium. Cells were washed twice in cold 1x PBS and lysed with a SDS-based lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1). Sonication of lysates was performed on ice using a Sonics and Materials Vibra-Cell sonicator with a 3-mm tip set at 30% maximum power with 10 s pulses and a 30s rest interval between every 5 pulses. Sonication was conducted to solubilize chromatin and fragment chromatin to a size of 500-1000 bp. Chromatin was pre-cleared with salmon-sperm DNA/protein A-agaraose for 1 h . Pre-cleared chromatin samples were incubated with 5 μl of pan-AcH4 (Millipore) or normal rabbit serum (Santa Cruz) overnight with agitation at 4 °C. Salmon sperm DNA protein A-agarose (Millipore) was added and allowed to bind for 60 min at 4 °C before extensive washing. Samples were eluted in 1% SDS, 0.1M NaHCO3 and formaldehyde cross-links were reversed overnight at 65 °C. ChIP samples were then subjected to proteinase K digestion and purified using the QIAquick PCR purification kit (QIAGEN) into 10 mM Tris, pH 8.5.
|
| Label |
Cy5
|
| Label protocol |
ChIP samples were amplified using the whole genome amplification kit (Sigma) and labelled according to maufacturer's ChIP-chip labeling recommendations (Agilent Tecnologies). Pan-AcH4 and corresponding normal rabbit serum ChIP samples were labelled with cyanine 5-dUTP and cyanine 3-dUTP respectively with the Agilent Genomic DNA labeling kit PLUS.
|
| |
|
| Channel 2 |
| Source name |
Human Aortic Vascular Smooth Muscle Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HuAoVSMC
antibody: rabbit serum
tag: normal rabbit serum control ChIP
|
| Growth protocol |
HuAoVSMC (ScienCell; https://www.sciencellonline.com/human-aortic-smooth-muscle-cells.html) were cultured according to provider's instructions using their recommended complete human smooth muscle cell medium. All biological replicates were used at passage 3-4.
See protocols in SAMPLES
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was conducted using the Chromatin Immunoprecipitation kit (Millipore, 17-295) according to the manufacturer's instructions. Briefly, 1x 106 cells were used per ChIP reaction. Cultured cells were fixed in 1% formaldehyde at 37 °C in 5% CO2 for 10 minutes by adding formaldehyde directly to the medium. Cells were washed twice in cold 1x PBS and lysed with a SDS-based lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1). Sonication of lysates was performed on ice using a Sonics and Materials Vibra-Cell sonicator with a 3-mm tip set at 30% maximum power with 10 s pulses and a 30s rest interval between every 5 pulses. Sonication was conducted to solubilize chromatin and fragment chromatin to a size of 500-1000 bp. Chromatin was pre-cleared with salmon-sperm DNA/protein A-agaraose for 1 h . Pre-cleared chromatin samples were incubated with 5 μl of pan-AcH4 (Millipore) or normal rabbit serum (Santa Cruz) overnight with agitation at 4 °C. Salmon sperm DNA protein A-agarose (Millipore) was added and allowed to bind for 60 min at 4 °C before extensive washing. Samples were eluted in 1% SDS, 0.1M NaHCO3 and formaldehyde cross-links were reversed overnight at 65 °C. ChIP samples were then subjected to proteinase K digestion and purified using the QIAquick PCR purification kit (QIAGEN) into 10 mM Tris, pH 8.5.
|
| Label |
Cy3
|
| Label protocol |
ChIP samples were amplified using the whole genome amplification kit (Sigma) and labelled according to maufacturer's ChIP-chip labeling recommendations (Agilent Tecnologies). Pan-AcH4 and corresponding normal rabbit serum ChIP samples were labelled with cyanine 5-dUTP and cyanine 3-dUTP respectively with the Agilent Genomic DNA labeling kit PLUS.
|
| |
|
| |
| Hybridization protocol |
Cy5-labelled pan-AcH4 and corresponding Cy3-labelled rabbit serum ChIP samples were hybridized to custom 1M tiling arrays in accordance to manufacturer's ChIP-chip hybridization recommendations using the Agilent microarray hybridization chamber system.
|
| Scan protocol |
Microarrays were washed and scanned using the Agilent G2565C DNA Scanner.
|
| Description |
HuAoVSMC pan-AcH4 versus rabbit serum ChIP-chip analysis using the custom 1M ultra high resolution tiling array
|
| Data processing |
Agilent Feature Extraction software (Agilent) was used to analyze acquired array images. Quantified data files from Agilent Feature Extraction software (Agilent) were then collected into a single directory. Each file contained sense and corresponding antisense tiled probes to genomic regions surrounding a predefined list of genes with either EC-enriched expression, roles in EC physiology, or roles as control genes. Mean expression measurements (cy5 and cy3 channels) for each of the probes were extracted and sorted into an ascending chromosomal order as defined by the mid-point of the probe location in a format acceptable for input into the Tilemap command line software using custom scripts written in PERL. Preprocessing of the data consisted of quantile normalization followed by taking the log base 2. Bar files were then created for input to the Tilemapv2 program to find pulled down fragment peaks using the moving average (MA) mode. The MA algorithm uses a modified t-statistic averaged over a sliding 400 bp window of adjacent probes. False Discovery Rate (FDR) calculations are based on a left tail distribution estimate of the MA test statistics. We used an MA cutoff of 3.0 for peak selection. Based on the average probe spacing on the custom Agilent arrays, a window size of 3 was chosen for the tilemap configuration parameter file. Various choices for the configuration of tilemap were tested during the course of experiments to optimize peak finding using both real and simulated data.
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| |
|
| Submission date |
Jan 23, 2017 |
| Last update date |
Jan 19, 2018 |
| Contact name |
Philip A Marsden |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Toronto
|
| Department |
Medicine
|
| Street address |
209 Victoria Street, Room 522
|
| City |
Toronto |
| ZIP/Postal code |
M5B 1C6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL22952 |
| Series (1) |
| GSE93962 |
Pan-AcH4 ChIP-chip with High Resolution Tiling Arrays on HUVECs and HuAoVSMCs |
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