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| Status |
Public on May 06, 2013 |
| Title |
PGT44 |
| Sample type |
SRA |
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| Source name |
embryos
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain: w1118
developmental stage: 0-2 hour embryos
note: In this sample 10ng of human sperm DNA was spiked in
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| Growth protocol |
Drosophila embryos were collected by placing 1-3 day old flies in a large embryo collection cages, with a 10cm grape agar dish. After females had been in the cage overnight (to synchronize egglaying) embryos were collected every 2 hours. Embryos were brushed off agar plates using PBS, dechorionized for 2 minutes in 100% bleach and flash frozen in liquid nitrogen until genomic DNA extraction.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To extract genomic DNA, embryos were homogenized in 2X PK Buffer (200 mM Tris-HCl, pH 7.5, 25 mM EDTA, pH 8, 300 mM NaCl, 2% SDS) followed by phenol choloroform extraction and ethanol precipitation. Samples were then trated with RNAseA and purified again by phenol-chloroform and ethanol precipitation. To make bisulfite converted libraries, 1 ug of genomic DNA was sonicated with a Bioruptor using 3 cycles of 7 minutes (30 seconds on and 30 seconds off on high). Ice water was replaced after each cycle. Sonicated DNA was incubated with a mixture of T4 DNA polymerase (NEB), T4 Polynucleotide kinase (NEB) and standard Taq Polymerase (Roche) in 1X T4 DNA ligase buffer with 10mm ATP (NEB). Methylated illumina Paired End adaptors were ligated using Rapid T4 DNA Ligase (Roche). Adaptors and ligase were added directly to the previous reaction. Ligated fragments were purified using Zymo Clean and Concentrator -5. Bisulfite conversion was performed using the EZ DNA Methylation kit (Zymo), following the manufacturer's instructions. Library was PCR amplified using Roche HIgh Fidelity Plus enzyme, and 2ul bisulfite converted DNA template. 2 PCR reactions were done in parallel and pooled. Final library was purified using Zymo clean and concentrate -25.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
In this sample 10ng of human sperm DNA was spiked in
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| Data processing |
Illumina Casava1.8.1 software used for basecalling.
Preprocessing, read trimming: shore 0.6.2
Preprocessing, quality filtering: shore 0.6.2
BS-mapping: bsmap 2.0
computation of methylation ratios: methratio.py (part of bsmap package)
Genome_build: dm3
Supplementary_files_format_and_content: Bedgraph-format containing the genomic methylation ratios of the respective sample
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| Submission date |
Aug 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Guenter Raddatz |
| Organization name |
German Center for Cancer Research
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| Street address |
Im Neuenheimer Feld 580
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| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE39996 |
Dnmt2-dependent methylomes lack biologically relevant DNA methylation patterns [Drosophila melanogaster] |
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| Relations |
| SRA |
SRX174810 |
| BioSample |
SAMN01109517 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM983094_PGT44.bedgraph.gz |
293.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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