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| Status |
Public on Dec 03, 2018 |
| Title |
Sperm Donor1 Bisulfite Sequencing |
| Sample type |
SRA |
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| Source name |
Sperm Donor 1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: sperm
age: 32
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| Treatment protocol |
Sperm pellet samples were washed in 1X PBS and centrifuged at 10,000 G for 3 min.
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| Growth protocol |
The two sperm donor samples were obtained from men of known fertility attending the University of Utah Andrology laboratory, consented for research. Samples were collected after 2-5 days abstinence and subjected to a density gradient to purify viable, motile, mature sperm.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sperm DNA was extracted as previously described (Hossain et al., 1997; Bahnak et al., 1998). Three micrograms of genomic DNA were sheared using a COVARIS Adaptive Focused Accoustics S-series system (Woburn, Massachusetts 01801-1721). Sheared DNA was purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA 91355). DNA concentration was measured and unmethylated lambda DNA was added at a concentration to represent 1% of the DNA mass of each sample. The lambda DNA served as an internal control for conversion efficiency. (Promega Madison, WI 53711). Fully methylated paired-end adapters from the TruSeq DNA Sample Prep Kit were ligated to sheared DNA (following the Illumina library preparation protocol). After the adapter addition, samples were cleaned using a QIAquick PCR purification kit (Qiagen, Valencia, CA 91355) and subjected to sodium bisulfite conversion. Sodium bisulfite treatment was done using the EpiTect Bisulfite Kit (Qiagen, Valencia, CA 91355) with modified PCR conditions to ensure ~99.98% conversion efficiency: 98°C 10 min, 60°C 25 min, 98°C 10 min, 60°C 85 min, 98°C 10 min, 60°C 175 min, 98°C 10 min, 60°C 120 min, 20°C storage for the next step in library prep. Following cleanup of bisulfite-treated DNA, sequencing libraries were enriched by 16 cycles of PCR amplification using Agilent PfuTurbo Cx HotStart DNA Polymerase (Agilent, Santa Clara, CA 95051) to complete the library construction process. DNA fragments in the size range of 200-300bp were gel purified. Libraries were quality controlled by running the final library on an Agilent DNA 1000 Bioanalyzer chip DNA Chip and by qPCR using the KapaBiosystems KAPA Library Quant Kit for Illumina sequencing libraries. Libraries were sequenced on a 101 cycle paired-end sequencing run on an Illumina HiSeq 2000.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
7795X1
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| Data processing |
Basecalls were performed using CASAVA version 1.7.
Reads were aligned using Novoalign V2.07.03 with parameters -rRandom -t120 -h120 -b2 to an index containing the hg18 genome (UCSC), Lambda, PhiX, and Illumina Adapter sequences. The index was made using novoindex with parameter -b.
Alignments were filtered and converted to binary PointData with NovoalignBisulfiteParser (USeq package, version 7.6.4) using the default thresholds for quality score and alignment score and the option -u to get unique alignments.
Filtered alignments (PointData) were analyzed to determine methylation statistics, summary plots, and to determine differentially methylated regions with BisStat and BisSeq (USeq) using the default parameters.
Genome_build: hg18
Supplementary_files_format_and_content: Bed files contain hypermethylated or hypomethylated regions in merged sperm donor data.
'GSE40178_D1D2_HypermethylatedLoci_FractionMethylation>0.8%_SerializedWindowObjectsMin0.8Max1.0.bed' and 'GSE40178_D1D2_HypomethylatedLoci_Less20%FractionMethylation_SerializedWindowObjectsMin1.0E-4Max0.2.bed' are linked to the Series record as supplementary files.
Supplementary_files_format_and_content: Bar files contain tracks for visualization of methylation data in the Integrated Genome Browser (IGB) genome browser.
'GSE40178_BisStatFullCoverageGraphs.tar', 'GSE40178_BisStat_GCContexts.tar', and 'GSE40178_NBP.tar' are linked to the Series record as supplementary files.
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| Submission date |
Aug 16, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew J Oler |
| E-mail(s) |
andrew.oler@nih.gov
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| Organization name |
NIAID/NIH
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| Department |
Bioinformatics and Computational Biosciences Branch (BCBB)
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| Lab |
Computational Biology Section
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| Street address |
31 Center Drive, Room 3B62E
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE40178 |
Human Sperm Epigenomes and Transcriptomes Reveal Novel Features of Enhancers, Sex Chromosomes, piRNAs, Gametogenesis, and Inherited Small RNAs (Bisulfite-Seq) |
| GSE40196 |
Human Sperm Epigenomes and Transcriptomes Reveal Novel Features of Enhancers, Sex Chromosomes, piRNAs, Gametogenesis, and Inherited Small RNAs |
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| Relations |
| SRA |
SRX178602 |
| BioSample |
SAMN01120622 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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