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| Status |
Public on Dec 01, 2010 |
| Title |
SCC4 HPV(-), MeDIP-ChIP, array F |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
MeDIP-ChIP DNA from SCC4, HPV(-)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SCC4
cell type: head and neck squamous cell carcinoma (HNSCC)
hpv status: negative
tissue derivation: tongue
age: 47
gender: female
antibody: anti-5-methyl-cytosine
antibody vendor: Capralogics
antibody catalog #: P00704
antibody lot #: P704020607
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| Growth protocol |
HPV(+) and HPV(-) SCC cell lines were cultured to near confluence in ventilated 75cm2 cell culture flasks in DMEM (Gibco) supplemented with 20% fetal bovine serum (FBS) and prophylactic antibiotics. At 90-95% confluence, the cells were photographed through a 20x objective lens, and then harvested with Trypsin/EDTA. Total cell counts for each flask were obtained using a Coulter Particle Counter (Beckman Coulter). Trypsin was neutralized with culture media containing 2% FBS, and cell pellets were washed with sterile phosphate-buffered saline (PBS), pH 7.4, prior to digestion for nucleic acid isolation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using a modified protocol for the QiaAmp DNA Mini Kit for Blood and Body Fluids (Qiagen, Valencia, CA). In brief, ≤5 million cells were suspended in 200uL of PBS to which 20uL of Proteinase K (Qiagen) was added. A 5mm stainless steel bead was added to each tube and cells were simultaneously disrupted and homogenized with 2 bursts at 15Hz, 20 seconds each, in a TissueLyser II (Qiagen). Following homogenization, 20uL of RNase A (Qiagen) was added to each tube. After the addition of RNase A, DNA isolation was carried out as per manufacturer’s instructions for processing blood and body fluids with spin column technology. Genomic DNA was eluted in 100uL final volume of nuclease-free water, pre-warmed to 40°C. Four 25uL elutions were performed, each of which were incubated on the spin column membrane for 5 minutes before eluting.
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| Label |
biotin
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| Label protocol |
Methylated DNA IP-on-Chip was performed with GenPathway (San Diego, CA) using the GeneChip Human Tiling 2.0R Array Set (Affymetrix, Santa Clara, CA). Following preclearing with protein G agarose beads (Invitrogen), DNA was denatured and immunoprecipitated using an anti-5-methyl-cytosine antibody (Capralogics, P00704, Lot# P704020607), and incubated overnight at 4C, with protein G agarose beads. An aliquot of each precipitated sample was used for QPCR to confirm the enrichment of methylated regions, using a GenPathway proprietary method. Immunoprecipitated and input DNAs (mock-IP'ed SCC4, SCC47, SCC74A, and CaSki cell lines) were amplified, and QPCR again verified enrichment of methylated regions in IP'ed DNA. Amplified immunoprecipitated and input DNAs were labeled according to Affymetrix procedures.
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| Channel 2 |
| Source name |
Input DNA, mock IP
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| Organism |
Homo sapiens |
| Characteristics |
cell line: UM-SCC-4, UM-SCC-47, UM-SCC-74A, and CaSki
antibody: none
|
| Growth protocol |
HPV(+) and HPV(-) SCC cell lines were cultured to near confluence in ventilated 75cm2 cell culture flasks in DMEM (Gibco) supplemented with 20% fetal bovine serum (FBS) and prophylactic antibiotics. At 90-95% confluence, the cells were photographed through a 20x objective lens, and then harvested with Trypsin/EDTA. Total cell counts for each flask were obtained using a Coulter Particle Counter (Beckman Coulter). Trypsin was neutralized with culture media containing 2% FBS, and cell pellets were washed with sterile phosphate-buffered saline (PBS), pH 7.4, prior to digestion for nucleic acid isolation.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using a modified protocol for the QiaAmp DNA Mini Kit for Blood and Body Fluids (Qiagen, Valencia, CA). In brief, ≤5 million cells were suspended in 200uL of PBS to which 20uL of Proteinase K (Qiagen) was added. A 5mm stainless steel bead was added to each tube and cells were simultaneously disrupted and homogenized with 2 bursts at 15Hz, 20 seconds each, in a TissueLyser II (Qiagen). Following homogenization, 20uL of RNase A (Qiagen) was added to each tube. After the addition of RNase A, DNA isolation was carried out as per manufacturer’s instructions for processing blood and body fluids with spin column technology. Genomic DNA was eluted in 100uL final volume of nuclease-free water, pre-warmed to 40°C. Four 25uL elutions were performed, each of which were incubated on the spin column membrane for 5 minutes before eluting.
|
| Label |
biotin
|
| Label protocol |
Methylated DNA IP-on-Chip was performed with GenPathway (San Diego, CA) using the GeneChip Human Tiling 2.0R Array Set (Affymetrix, Santa Clara, CA). Following preclearing with protein G agarose beads (Invitrogen), DNA was denatured and immunoprecipitated using an anti-5-methyl-cytosine antibody (Capralogics, P00704, Lot# P704020607), and incubated overnight at 4C, with protein G agarose beads. An aliquot of each precipitated sample was used for QPCR to confirm the enrichment of methylated regions, using a GenPathway proprietary method. Immunoprecipitated and input DNAs (mock-IP'ed SCC4, SCC47, SCC74A, and CaSki cell lines) were amplified, and QPCR again verified enrichment of methylated regions in IP'ed DNA. Amplified immunoprecipitated and input DNAs were labeled according to Affymetrix procedures.
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| Hybridization protocol |
Hybridization mixes were prepared using the Affymetrix GeneChip Hybridization Wash and Stain Kit (Part 900720). Hybridization was at 45oC for 16-18 hours in an Affymetrix GeneChip Hybridization Oven.
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| Scan protocol |
Scanning was performed with GenPathway (San Diego, CA) using the GeneChIP Scanner 3000 7G.
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| Description |
SCC4 HPV(-)
Methylated DNA, which is immunoprecipitated using an antibody against 5-methyl-cytosine, is compared against input DNA.
The input DNA sample is a mixture of the mock-immunoprecipitated DNA from the four cell lines.
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| Data processing |
The Affymetrix Human Tiling 2.0R Array Set is a set of 7 tiling arrays divided by chromosome, and consisting of a total of more than 40 million 25-mer oligos. Raw data were read into R using the AffyTiling package and were quantile normalized. Oligos were annotated to CpG islands and genes using the UCSC CpG islands and known genes tracks, respectively. Testing for methylated and differentially methylated regions was performed using an empirical Bayes approach with moderated variance in ANOVA. This method takes into account the dependency of variance on intensity level, with variance tending to decrease with increasing intensity. The estimates of fold change over input and between HPV(+) and HPV(-) cells were smoothed using a moving window average of 175 bp length.
The BED and input DNA CEL files are linked to the Series GSE24090 record as supplementary files.
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| Submission date |
Sep 12, 2010 |
| Last update date |
Sep 16, 2011 |
| Contact name |
Jung Kim |
| E-mail(s) |
junghk@umich.edu
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| Organization name |
University of Michigan
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| Street address |
1420 Washington Heights
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL4915 |
| Series (2) |
| GSE24090 |
Genome-wide methylation analysis in HPV(-) and HPV(+) squamous cell carcinoma cell lines |
| GSE24091 |
Expression and methylation analyses of HPV(-) and HPV(+) squamous cell carcinoma cell lines |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM593038_5me_SCC4_ArrayF_091015.CEL.gz |
24.1 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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