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| Status |
Public on Jul 07, 2020 |
| Title |
Medaka Dnmt1 Mut2 |
| Sample type |
SRA |
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| Source name |
dnmt1 mutant rep2
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| Organism |
Oryzias latipes |
| Characteristics |
tissue: whole embryo
genotype: dnmt1-/-
strain: cab
surface phenotype: n/a
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from 3 dnmt1+/+ and 3 dnmt1-/- medaka at 18 dpf using DNeasy blood and tissue kit (Qiagen).
150ng of DNA was used for the bisulfite reaction using the EZ DNA methylation kit (Zymo Research) and the bisulfite-treated DNA was recovered in 15ml of elution buffer. 1ml of bisulfite-treated DNA solution was used for 30 cycles of PCR amplification with the indicated bisulfite primers in a reaction volume of 20ml; 1ml of the first PCR reaction was used for a nested PCR reaction with P5 and P7 adaptor-attached bisulfite primers. After the nested PCR reactions, amplicons were purified using PCR purification kit (Qiagen) and qualified and quantified by gel electrophoresis, the High Sensitivity DNA kit (Agilent) for the Agilent Bioanalyzer and the Qubit dsDNA HS assay kit (Invitrogen). The eight amplicons derived from the same original source were mixed at 1ng/ml each and 15ml of the mixture were amplified using P5 and P7 primers attached to barcodes using NEBNext DNA library prep kit (New England Biolabs), following the manufacturer«s protocol. The amplicons were purified using AMPure XP (Beckman Coulter) and qualified and quantified using Bioanalyzer and Qubit dsDNA HS assay kit. Finally, all amplicons were mixed at 1ng/ml each and 5 ml of mixed amplicons were used for sequencing in paired-end 150bp mode on 1 lane of MiSeq Sequencing System (Illlumina).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Basecalling was performed with Illumina MiSeq Control Software/RTA version 2.6.2.1.
Raw sequencing reads were trimmed with cutadapt version 1.9.1 (Martin, 2011), removing the first two 5'-most nucleotides and Illumina adapter sequences. Base quality trimming was performed using prinseq lite version 0.20.4 .
Bisulfite-specific mapping to bisulfite-converted MEDAKA1.72 genome was performed with Bismark v0.14.6 and Bowtie2 v2.2.8 in non-directional mode allowing for dovetail alignments.
Samtools sorted and indexed bams were used for methylation bias calculation (MethylDackel v0.1.7) and read count extraction in target regions with methylCtools version 0.9.4(Hovestadt et al., 2014), using MAPQ threshold 10, SNP detection, counting one of two overlapping mates, skipping 5 nucleotides from read ends, and zero-padding.
Data postprocessing was performed in R version 3.2.3. Raw methylation values were set to NA for CpG positions with at least 0.25 SNP allelic frequency as well as for positions with aggregate coverage of less than 10 reads. Single CpG methylation values were aggregated per target genomic interval as mean values.
Genome_build: MEDAKA1.72
Supplementary_files_format_and_content: Single CpG as well as mean methylation values in target genomic intervals are summarized in corresponding tab-separated tables for all samples.
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| Submission date |
May 08, 2017 |
| Last update date |
Jul 07, 2020 |
| Contact name |
Thomas Boehm |
| E-mail(s) |
boehm@ie-freiburg.mpg.de
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| Organization name |
MPI-IE Freiburg
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| Department |
Immunology
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| Street address |
Stübeweg 51
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| City |
Freiburg im Breisgau |
| ZIP/Postal code |
79108 |
| Country |
Germany |
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| Platform ID |
GPL23444 |
| Series (2) |
| GSE98646 |
Structural basis of epigenetic protection of mouse lymphoid progenitor cells by Dnmt1 (Medaka) |
| GSE98648 |
Structural basis of epigenetic protection of mouse lymphoid progenitor cells by Dnmt1 |
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| Relations |
| BioSample |
SAMN06915130 |
| SRA |
SRX2789123 |
| 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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