|
| Status |
Public on Jun 15, 2009 |
| Title |
InputSpermD1 and Histone-MNAseSpermD1, Replica 1, Slide 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Sperm Patient D1
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: sperm
|
| Growth protocol |
Sperm samples were obtained from three known fertile donors attending the University of Utah Andrology laboratory, consented for research. Primary human fibroblast cells were obtained from Lonza (CC2511) and were cultured in DMEM containing 10% FBS and supplemented with penicillin and streptomyocin (37°C and 5% CO2).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sperm samples were collected after 2-5 days abstinence and subjected to an ISolate density gradient (to purify viable, motile, mature sperm) and treated with somatic cell lysis buffer (0.1% SDS, 0.5% Triton X in DEPC H2O) for 20 min on ice to eliminate white blood cell contamination. Samples were centrifuged at 10,000 G for 3 min and the sperm pellet was resuspended in 1X PBS and used immediately for chromatin preparation. Chromatin was prepared as previously described by Zalenskaya et al Biochem Biophys Res Commun 279, 213-8 (2000) in the absence of crosslinking reagent. Sperm was treated with sequential and increasing MNase (10U-160U), and centrifuged to sediment protamine-associated DNA, releasing mononucleosomes. The pooled mononucleosomes were used for chromatin immunopreciptation (below), or the DNA extracted and gel purified (~140-155bp) for array analysis. All Chromatin IPs (ChIPs) were performed as previously described by Gordon et al Mol Cell Biol 27, 4058-69 (2007). All Data for CHIP modifications and histone variants were normalized to the three Histone replicates as input. Fibroblast chromatin was processed according to Zalenskaya et al 2000 and ChIPed according to Gordon et al 2007.
|
| Label |
Cy3
|
| Label protocol |
PCR-amplified DNA samples (2-3 µg) derived from chromatin immunoprecipitation reactions were labeled using the Agilent Genomic DNA Labeling Kit PLUS. The labeling kit uses random primers and the exo-Klenow fragment to label DNA through incorporation of fluorescently labeled nucleotides (Cy3-dUTP or Cy5-dUTP). Following termination of the labeling reaction, fluorescently labeled DNA molecules were purified by isopropanol precipitation. Precipitated pellets were dried and then rehydrated in distilled water. The concentration of purified samples was determined using a NanoDrop ND-1000 spectrophotometer. A fraction of the labeled DNA (100 ng) was run on an Agilent BioAnalyzer to validate the size distribution of the labeled DNA molecules.
|
| |
|
| Channel 2 |
| Source name |
Sperm Patient D1
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: sperm
|
| Growth protocol |
Sperm samples were obtained from three known fertile donors attending the University of Utah Andrology laboratory, consented for research. Primary human fibroblast cells were obtained from Lonza (CC2511) and were cultured in DMEM containing 10% FBS and supplemented with penicillin and streptomyocin (37°C and 5% CO2).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sperm samples were collected after 2-5 days abstinence and subjected to an ISolate density gradient (to purify viable, motile, mature sperm) and treated with somatic cell lysis buffer (0.1% SDS, 0.5% Triton X in DEPC H2O) for 20 min on ice to eliminate white blood cell contamination. Samples were centrifuged at 10,000 G for 3 min and the sperm pellet was resuspended in 1X PBS and used immediately for chromatin preparation. Chromatin was prepared as previously described by Zalenskaya et al Biochem Biophys Res Commun 279, 213-8 (2000) in the absence of crosslinking reagent. Sperm was treated with sequential and increasing MNase (10U-160U), and centrifuged to sediment protamine-associated DNA, releasing mononucleosomes. The pooled mononucleosomes were used for chromatin immunopreciptation (below), or the DNA extracted and gel purified (~140-155bp) for array analysis. All Chromatin IPs (ChIPs) were performed as previously described by Gordon et al Mol Cell Biol 27, 4058-69 (2007). All Data for CHIP modifications and histone variants were normalized to the three Histone replicates as input. Fibroblast chromatin was processed according to Zalenskaya et al 2000 and ChIPed according to Gordon et al 2007.
|
| Label |
Cy5
|
| Label protocol |
PCR-amplified DNA samples (2-3 µg) derived from chromatin immunoprecipitation reactions were labeled using the Agilent Genomic DNA Labeling Kit PLUS. The labeling kit uses random primers and the exo-Klenow fragment to label DNA through incorporation of fluorescently labeled nucleotides (Cy3-dUTP or Cy5-dUTP). Following termination of the labeling reaction, fluorescently labeled DNA molecules were purified by isopropanol precipitation. Precipitated pellets were dried and then rehydrated in distilled water. The concentration of purified samples was determined using a NanoDrop ND-1000 spectrophotometer. A fraction of the labeled DNA (100 ng) was run on an Agilent BioAnalyzer to validate the size distribution of the labeled DNA molecules.
|
| |
|
| |
| Hybridization protocol |
Fluorescently labeled DNA samples were heat denatured after being combined with cot-1 DNA, Agilent aCGH blocking agent and Agilent Hi-RPM hybridization solution. Microarray hybridizations were performed using Agilent SureHyb Hybridization chambers. Hybridization chambers were loaded onto a rotisserie in an Agilent Hybridization Oven and were incubated at 65ºC for 40 hours with a rotational speed of 20 rpm. Following incubation, the microarray slide was washed for 5 minute in aCGH/ChIP-on-chip Wash Buffer 1 (0.5X SSPE, 0.005% N-lauroylsarcosine; room temperature) and 5 minute in aCGH/ChIP-on-chip Wash Buffer 2 (0.1X SSPE, 0.005% N-lauroylsarcosine; 31ºC). Microarray slides were briefly dipped in a solution of acetonitrile and dried.
|
| Scan protocol |
Microarray slides were scanned in an Agilent Technologies G2505B Microarray Scanner at 5 µm resolution. The scanner performs simultaneous detection of Cyanine-3 and Cyanine-5 signal on the hybridized slide. Data captured from the scanned microarray image is saved as a TIF file.
|
| Description |
histone bound DNA enrichment via MNase digestion and nucleosome isolation, replica 1
|
| Data processing |
The Tiling Microarray Analysis Tools 2 package (http://timat2.sourceforge.net) was used to quantile normalize both treatment and control samples, median scale, and window scan (150bp) the tiled gene promoters. Rank lists (best to worst) of enriched regions/ intervals were generated by setting log2 ratio thresholds that generated the top 100, 200, 400, 800, 1600, 3200, and 6400 regions.
The enriched regions are listed in the '*EnrichedRegions.xls' files, available from the GSE15701 record.
The files containing oligo ratio data (log2(AveTreatment/AveControl)) are in the '*OligoRatioData.zip' folders, also available from the GSE15701 record.
All coordinates are relative to Hg18/ NCBI 36.1/ H_sapiens_Mar_2006.
|
| |
|
| Submission date |
May 13, 2009 |
| Last update date |
Jun 15, 2009 |
| Contact name |
Bradley R. Cairns |
| Organization name |
HHMI and Huntsman Cancer Institute
|
| Department |
Oncological Sciences
|
| Street address |
2000 Circle of Hope
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84102 |
| Country |
USA |
| |
|
| Platform ID |
GPL4125 |
| Series (2) |
| GSE15594 |
Distinctive Chromatin in Human Sperm Packages Genes that Guide Embryo Development |
| GSE15701 |
Distinctive Chromatin in Human Sperm Packages Genes that Guide Embryo Development: ChIP-chip |
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