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| Status |
Public on Dec 15, 2022 |
| Title |
Dnmt3ab WT, E7.5, Epi, 2 (PBAT) |
| Sample type |
SRA |
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| Source name |
Epiblast
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| Organism |
Mus |
| Characteristics |
cell type: Epiblast
strain: C57BL6
genotype: WT
age: E7.5
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Natural timed matings between heterozygous females and males were used for Dnmt3l, Dnmt3a, Dnmt3b and Dnmt3a/3b KO strains to generate KO and WT embryos. E7.5 epiblast, extra-embryonic ectoderm (ExE) and ectoplacental cone (EPC) for each embryo were manually separated. Single E7.5 epiblast and ExE samples were individually frozen in 10µL of buffer RLT Plus (Qiagen) for RNA-seq or PBAT; the corresponding EPC was used for genotyping. Cells were lysed with 0.5% SDS at 37°C for 1 hour and bisulphite converted using the Imprint DNA Modification kit (Sigma). Bisulphite-converted DNA was purified using the EZ DNA Methylation Direct kit, as directed (Zymo Research). Using a biotin-conjugated adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences (9N), first-strand synthesis was performed using Klenow Exo- enzyme (New England Biolabs). Following exonuclease (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific), second-strand synthesis was performed. Libraries were amplified and indexed using 10 PCR cycles with Phusion High-Fidelity DNA polymerase (New England Biolabs). Libraries were multiplexed for 75-bp paired-end sequencing on an Illumina NextSeq500.
Lysed cells were bisulphite converted using the Imprint DNA Modification kit (Sigma). Bisulphite-converted DNA was purified using the EZ DNA Methylation Direct kit, as directed (Zymo Research). Using a biotin-conjugated adaptor containing standard Illumina adaptor sequences and 9 bp of random sequences (9N), first-strand synthesis was performed using Klenow Exo- enzyme (New England Biolabs). Following exonuclease (New England Biolabs) treatment and binding to Dynabeads M-280 Streptavidin beads (Thermo Fisher Scientific), second-strand synthesis was performed. Libraries were amplified and indexed using 10 PCR cycles with Phusion High-Fidelity DNA polymerase (New England Biolabs). Libraries were multiplexed for 75-bp paired-end sequencing on an Illumina NextSeq500.
PBAT (BS-seq)
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Fastq sequence files were quality and adaptor trimmed with trim galore v0.4.2 using default parameters. For PBAT libraries, the -clip option was used to remove the bases covered by the PBAT primer.
Mapping and methylation calling of bisulphite-seq data was performed using Bismark v0.16.3 in PBAT mode against the mouse GRCm38 genome assembly. Trimmed reads were first aligned to the genome in paired-end mode to be able to detect and discard overlapping parts of the reads while writing out unmapped singleton reads; in a second step remaining singleton reads were aligned in single-end mode. Alignments were carried out with Bismark44 with the following set of parameters: a) paired-end mode: --pbat; b) single-end mode for Read 1: --pbat; c) single-end mode for Read 2: defaults. Reads were then deduplicated with deduplicate_bismark selecting a random alignment for positions that were covered more than once. Following methylation extraction, CpG context files from PE and SE runs were used to generate a single coverage file (the "Dirty Harry" procedure).
Assembly: GRCm38
Supplementary_files_format_and_content: The Bismark CpG coverage report is tab-delimited, uses 1-based genomic coordinates for every covered cytosine position in the experiment and is in the following format: <chromosome> <start position> <end position> <methylation percentage> <count methylated> <count non-methylated>
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| Submission date |
May 18, 2022 |
| Last update date |
Dec 15, 2022 |
| Contact name |
Laura Biggins |
| E-mail(s) |
[email protected]
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| Organization name |
The Babraham Institute
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| Department |
Bioinformatics
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| Street address |
Babraham Research Campus
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| City |
Cambridge |
| ZIP/Postal code |
CB22 3AT |
| Country |
United Kingdom |
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| Platform ID |
GPL23642 |
| Series (2) |
| GSE203292 |
Mechanisms and function of de novo DNA methylation in placental development [PBAT] |
| GSE203462 |
Mechanisms and function of de novo DNA methylation in placental development |
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| Relations |
| BioSample |
SAMN28527690 |
| SRA |
SRX15328911 |
| 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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