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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 01, 2023 |
| Title |
3-X-TOP_Illumina_mESC_deep |
| Sample type |
SRA |
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| Source name |
Embryo blastocyst
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| Organism |
Mus musculus |
| Characteristics |
tissue: Embryo blastocyst
cell line: E14TG2a
cell type: Embryonic stem cells
genotype: WT
treatment: None
modification: 5-hydroxymethylcytosine, GC and unmodified CG sites
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| Treatment protocol |
Differentiation of mouse ESCs to neurons was performed, as described in Bibel et al., 2007.
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| Growth protocol |
ESCs were cultured on feeder-free 0.15% gelatin-coated plates in DMEM medium supplemented with 15% embryonic stem-cell FBS, 50 µg/ml penicillin–streptomycin mix, 2 mM L-glutamine, 1 × non-essential amino acids, 1 mM 2-mercaptoethanol (all from Gibco) and 1000 units/mL ESGRO mLIF (Milipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from nuclei was extracted by using standard phenol-chloroform extraction.
2-X-TOP Ion Torrent libraries - sonicated uCG- and GC- or 5hmC- and GC-azide labeled gDNA samples were used for the ligation of adaptors, as described in Gibas et al., 2020. After ligation, the alkyne-bearing oligodeoxyribonucleotide with biotin was attached to the azide moiety of the modified DNA. Biotinylated DNA was enriched with streptavidin-coupled magnetic beads and subsequently used in a DNA priming reaction from the tethered oligonucleotide. The primed DNA library was amplified, size-selected, and subjected to Ion Proton sequencing. 3-X-TOP Ion Torrent libraries - CG-, GC-alkyne, and 5hmC-azide labeled gDNA samples libraries were prepared using the same methodology as for 2-X-TOP Ion Torrent libraries, except that the biotinylated azide-containing and the biotinylated alkyne-containing oligonucleotides were click conjugated in two rounds. 3-X-TOP Illumina libraries - to CG-, GC-alkyne, and 5hmC-azide labeled gDNA samples adapters were attached by incubating the samples with hyperactive Tn5 transposase preloaded with Illumina-compatible sequencing adapters. The library was then prepared using the same methodology as for 3-X-TOP Ion Torrent library preparation (without the click product enrichment) and was subjected to Illumina sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
GC_table.tsv
hmC_table.tsv
uCG_table.tsv
regions_idGC.tsv
regions_idT.tsv
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| Data processing |
Raw data quality was evaluated using FASTQC. Reads were trimmed using cutadapt (v3.4) and in-house script.
Cleaned reads were mapped to the reference genome (mm10) using bwa (v0.7.17). Samtools (v1.9) were used to perform all manipulations with SAM/BAM files.
Each reads was assigned to a specific GC/CG target and coverage tables were generated using an in-house R (v.4.0.2) script. Open chromatin regions were identified using an in-house R script.
Assembly: mm10
Supplementary files format and content: Tab delimited table with target (5hmCG, uCG, GC) coverage per each sample.
Supplementary files format and content: Tab delimited table with identified coordinates of open chromatin regions.
Library strategy: TOP-Seq
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| Submission date |
May 08, 2023 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Kotryna Kvederaviciute |
| E-mail(s) |
kotryna.kvederaviciute@gmail.com
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| Organization name |
Vilnius University
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| Street address |
Universiteto str. 3
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| City |
Vilnuis |
| ZIP/Postal code |
01513 |
| Country |
Lithuania |
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| Platform ID |
GPL30172 |
| Series (2) |
| GSE231928 |
Unified analysis of DNA methylation, hydroxymethylation and chromatin accessibility through targeted DNA labeling [TOP-Seq] |
| GSE231929 |
Unified analysis of DNA methylation, hydroxymethylation and chromatin accessibility through targeted DNA labeling |
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| Relations |
| BioSample |
SAMN34999094 |
| SRA |
SRX20253313 |
| 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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