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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 07, 2018 |
| Title |
E10.5 PGC AbaSeq Replicate 2 |
| Sample type |
SRA |
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| Source name |
E10.5 PGC
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| Organism |
Mus musculus |
| Characteristics |
genetic background: MF1 x GOF18∆PE-EGFP
genotype: Wild type
age: E10.5
cell type: PGC
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total DNA was isolated from 10,000 sorted PGCs using the QIAamp DNA Micro Kit (Qiagen) with 0.8 µg of carrier RNA and following the “Isolating DNA from small samples of blood” protocol.
AbaSeq libraries for 5hmC profiling were constructed as previously described (23). In brief, genomic DNA was glucosylated with 400 nmol UDP-glucose and 10 units of T4-β-glucosyltransferase (NEB) in NEB buffer 4 overnight at 37°C and was purified afterward by phenol-chloroform extraction and ethanol precipitation. The glucosylated genomic DNA was then digested by four units of AbaSI enzyme (NEB) in NEB buffer 4 at room temperature (25°C) for 1 hr and the enzyme was inactivated at 65°C for 20 minute. Biotinylated P1 adapters were ligated onto the AbaSI digested DNA by incubating the digested DNA with 50 pmol of P1 and 2 μl of T4 DNA quick ligase (NEB) in the 1X quick ligation buffer in a total volume of 42 μl at room temperature for 30 min. Ligated DNA was then purified by AMPure XP beads (Beckman-Coulter) and was fragmented using a Covaris S2 sonicator (Covaris), following the manufacturer’s instructions. Sheared P1-ligated DNA was then captured by mixing with 12.5 μl of Dynabeads MyOne Streptavidin C1 beads (Life Technologies) according to the manufacturer’s specifications. End repair and dA-tailing were carried out on the beads by using the NEBNext End Repair Module (NEB) and the NEBNext dA-tailing Module (NEB) at 20°C and 37 °C respectively for 30 min. The beads were washed twice with a buffer containing 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, and 1 M NaCl between the two reactions and after dA-tailing. P2 adapters were ligated to the random sheared ends of the dA-tailed DNA using NEB T4 DNA quick ligase at room temperature for 30 min. After washing the DNA-bound beads as described above, the entire DNA was amplified using the Phusion DNA polymerase (NEB) with the addition of 300 nM forward primer (PCR_I) and 300 nM reverse primers (PCR_IIpe) for 16 cycles. The beads were then removed from the PCR and the library was purified using AMPure XP beads (Beckman-Coulter). Libraries were sequenced on the Illumina HiSeq 2000 instrument.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
AbaSeq of primordial germ cells
E10_AbaSeq_symmetric_noBlack_realNorm.bed
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| Data processing |
Library strategy: Aba-Seq
For the uniquely mappable part of the genome, raw sequencing reads were trimmed for adaptor sequences and low quality bases using Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The trimmed reads were mapped to the mouse genome (mm9, NCBI build 37) using Bowtie (version 0.12.8) with parameters -n 1 -l 25 --best --strata -m 1. Calling of 5hmC was based on the recognition sequence and cleavage pattern of the AbaSI enzyme (5′-CN11–13↓N9–10G-3′/3′-GN9–10↓N11–13C-5′) using custom Perl scripts. For assessing relative enrichment of 5hmC at repetitive elements and non-repetitive elements, AbaSeq alignments were divided into two groups: unique (single best alignment) and ambiguous (map to multiple locations with equal alignment score). Both groups were then mapped to the repetitive elements defined by the RepeatMasker track of mm9 (UCSC Genome Browser) separately. For quantification of relative 5hmC levels at symmetric CpGs in the uniquely mappable part of the genome, the number of counts per symmetric CpG for a given sample were normalized to the combined number of uniquely mapped and ambiguously mapped reads for a given library, and then further multiplied by a stage-specific normalization factor based on the mean 5hmC level for each stage as computed by LC/MS (E10.5 = 1.0; E11.5 = 1.13; E12.5F = 0.76; E12.5M = 1.0). All symmetric CpGs falling within genomic intervals blacklisted by the mouse (mm9) ENCODE project were excluded from all further downstream analysis.
Genome_build: mm9
Supplementary_files_format_and_content: BED file. Columns: <chromosome> <start position> <end position> <context> <normalized read counts> <strand>
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| Submission date |
Jan 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Hill |
| E-mail(s) |
peter.hill1@imperial.ac.uk
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| Organization name |
Imperial College London
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| Street address |
Kensington
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| City |
London |
| ZIP/Postal code |
SW7 2AZ |
| Country |
United Kingdom |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE76957 |
Epigenetic reprogramming enables the primordial germ cell-to-gonocyte transition (AbaSeq) |
| GSE76973 |
Epigenetic reprogramming enables the primordial germ cell-to-gonocyte transition |
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| Relations |
| BioSample |
SAMN04421018 |
| SRA |
SRX1534229 |
| 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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