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| Status |
Public on Nov 10, 2020 |
| Title |
3AC 3d dox Rep2 MBD2 seq |
| Sample type |
SRA |
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| Source name |
HEK293 cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
binding protein: GST-MBD2
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| Treatment protocol |
For induction of the ZnF-3AC construct, cell culture medium was supplemented with 1 µg/ml doxycyclin (Sigma-Aldirch) for three days
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) from frozen HEK293 pellets was isolated using the QIAmp DNA Mini Kit (Qiagen) according to manufacturer's protocol. gDNA was sonicated with a EpiShear probe sonicator (Active Motif) to obtain fragment sizes between 200 and 1000 bps. For pulldown, 1 µg of sheared gDNA was used with 8.75 µg GST-MBD2 in PB150 Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5 % Igepal CA-630, 2 mM DTT) and samples were incubated overnight at 4°C. The next day, 50 µl of Gluthathione Agarose beads (Macherey-Nagel) were washed three times with PB150 and the pulldown sample was added subsequently. After 2 h rotation at 4°C, DNA-GST-MBD2-beads complexes were washed three times with PB500 buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.5 % Igepal CA-630, 2 mM DTT) for 5 min. DNA was eluted twice with 150 µL PB2000 buffer at room temperature for 15 min. After pooling of elution fractions, the precipitated DNA was purified using the ChIP DNA Purification Kit (Active Motif) as described in the manufacturer's protocol.
Libraries for all ZnF-3AC related samples were constructed by the Max Planck-Genome-centre Cologne. In case of 3AC, libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to manufacturer's protocol.
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| Library strategy |
MBD-Seq |
| Library source |
genomic |
| Library selection |
MBD2 protein methyl-CpG binding domain |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
3AC_3dDox_5mC(0.66x).bigwig
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| Data processing |
Data were submitted in FASTQ format and all data processing steps were performed on the European Galaxy platform (usegalaxy.eu)
The quality of the reads was checked using the FastQC tool
Reads were mapped on human genome 19 (hg19/GRCh37) using bowtie2 in default settings
Reproducibility of replicates was confirmed and BAM-files of corresponding replicates were merged using the Merge BAM Files tool
Coverage files (bigwig) were generated using bamCoverage from DeepTools (25 bp bins) with RPKM normalization. Re-calibration of bigWig files was performed as follows: signals in CpG islands (CGIs) (obtained from UCSC Table Browser) were determined via multiBigwigSummary tool for each time point. The 1000 highest methylated CGIs at "no dox" were selected, the means in these CGIs were calculated for each time point and the ratios were used as scaling factor. BamCoverage was repeated in RPKM mode (25 bp bins) implying this scaling factor.
Genome_build: hg19/GRCh37
Supplementary_files_format_and_content: bigWig
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| Submission date |
Jan 27, 2020 |
| Last update date |
Nov 10, 2020 |
| Contact name |
Albert Jeltsch |
| E-mail(s) |
[email protected]
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| Phone |
+49 685 64390
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| Organization name |
University Stuttgart
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| Department |
Institute of Biochemistry and Technical Biochemistry
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| Street address |
Allmandring 31
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| City |
Stuttgart |
| ZIP/Postal code |
70569 |
| Country |
Germany |
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| Platform ID |
GPL21290 |
| Series (2) |
| GSE144331 |
Genome-wide investigation of the dynamic changes of epigenome modifications after DNA methylation editing [MBD-seq] |
| GSE144904 |
Genome-wide investigation of the dynamic changes of epigenome modifications after DNA methylation editing |
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| Relations |
| BioSample |
SAMN13930424 |
| SRA |
SRX7637906 |
| 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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