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| Status |
Public on Apr 24, 2020 |
| Title |
TTKO_cre2_d13 (SureSelect) |
| Sample type |
SRA |
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| Source name |
TET triple knockout mESCs
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
tissue: mESCs
genotype/variation: TET triple knockout (TTKO)
genotype/variation: Dnmt3a/3b catalytic dead
timepoint: day 13
replicate: replicate 2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
SureSelect enrichment and subsequent sequencing of bisulfite DNA was carried out as per the manufacturer’s instructions. Briefly, genomic DNA isolated from the time points above was sonicated down to 150-200 base pairs in size using a Covaris S220, followed by library construction. The libraries were then hybridized to probes (Mouse Methyl-Seq XT, 931052), bound to streptavidin beads, washed and bisulfite converted using the EZ DNA methylation Gold kit from Zymo (D5005). Bisulfite converted DNA libraries were then amplified and indexed, pooled and sequenced in 51bp paired-end mode using the Illumina HiSeq platform.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
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| Description |
Post-Cre transduction day 13
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| Data processing |
Alignment: For amplicon bisulfite samples, the last 100 base pairs were trimmed due to reduced sequencing quality using the preprocessReads function from the Bioconductor QuasR package with the truncateEndBases = 100 parameter. Amplicon sequences were then aligned to the mm9 build using Rbowtie in the QuasR package and the following parameters: genome="BSgenome.Mmusculus.UCSC.mm9", paired="fr", bisulfite="undir". For SureSelect samples, reads were aligned to the mm9 build using Rbowtie in the QuasR environment as for amplicon sequencing above except with the parameter bisulfie = "dir".
Methylation level determination: Methylation levels for CpGs in amplicons was determined using the AmpliconViews function from the R package AmpliconBiSeq with the parameters ‘conv = 80’ and exp.var = ‘90’. Methylation levels for SureSelect samples was calculated using the qMeth function in the QuasR package. For the SureSelect methylation data (Supplemental_Table4), read counts for replicates of the same time points were added together before determining methylation levels. Coverage columns for Supplemental_Table4 reflect summed counts for the triplicate experiments.
Filtering: A minimum of 100x coverage was required for all methylation calls in the amplicon bisulfite data. If coverage did not reach this threshold for any time point, the respective methylation level was set to NA. We further removed all CpGs with NA at day 0 or with more than one NA during the time course across all replicates. In addition, we filtered out one amplicon (chr7:149767665-149767784) containing 9 CpGs due to high variability in methylation calls. From a total of 588 CpGs initially quantified, this filtering procedure resulted in 405 CpGs. For SureSelect, a minimum of 50x coverage in all time points and replicates was used to filter CpGs for further analysis.
Rate determination: For methylation and demethylation rate determination, please see text.
Genome_build: mm9
Supplementary_files_format_and_content: Table files with CpG coordinates, methylation level, coverage, rate of methylation, rate of demethylation and tet activity (SureSelect only). For sample identication in Supplemental tables, there are 4 elements in the sample name that reflect the sample composition. Taking the sample name 'TTKO_cre1_d4_methylation' as an example, the TTKO refers to 'Tet Triple Knock-Out', while the 'cre1' refers to 'Cre transduction, replicate 1', the element 'd4' refers to 'Day 4 post transduction' and finally 'methylation' is the percent methylated alleles at that CpG. Other acronyms used are 'WT' for 'Wild-type', 'coverage' for number of reads covering that particular CpG in the sample, and 'mock' refers to the negative control in cre transduction. More specifically, 'm' refers to 'mock' and indicates addition of DMSO to the cells instead of Cre protein. The column 'MLE_kMe' reflects the rate of methylation while 'MLE_kDe' reflects the rate of demethylation (described in detail in the manuscript). The confidence column reflects the confidence level of rate inferrence. Lastly, the 'tag' column in Supplemental Table 2 and 3 reflects whether the CpG resides in an LMR (Low Methylated Region), FMR (Fully Methylated Region), UMR (Unmethylated Region), metIsland (Methylated CpG Island), NP-specific (Neural Progenitor specific) and ES-specific (Embyonic Stem cell specific) region. The designation 'Constitutive' reflects that the CpG resides in regions with the methylation state of interest in different cell types. In Supplemental Table 2, 'inferredInWTandTET' indicates rates inferred in wild-type and TTKO timecourse, while 'kDeWT_predicted' indicates predicted demethylation rates in wild-type cells using TTKO time course information and wild-type steady state measurements.
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| Submission date |
Apr 08, 2019 |
| Last update date |
Apr 24, 2020 |
| Contact name |
Dirk Schuebeler |
| Organization name |
Friedrich Miescher Institute for Biomedical Research
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE129470 |
A genome-scale map of DNA methylation turnover identifies site-specific dependencies of DNMT and Tet activity |
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| Relations |
| BioSample |
SAMN11358538 |
| SRA |
SRX5653820 |
| 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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