|
| Status |
Public on Jun 18, 2021 |
| Title |
MeDIP_P10_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
J1 mESC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
strain: J1
genotype: Dnmt3a + Dnmt3b KO
|
| Treatment protocol |
Passaged ten times after Dnmt1 shRNA reversal with transiently transfected Cre recombinase plasmid
|
| Growth protocol |
mES cells were cultured in Knock Out DMEM media (Gibco) containing 15% FBS, 1% PEN/STREP, 1% glutamine, 1% NEAA, and 105 U LIF. For ES cell maintenance, dishes were coated with 0.2% gelatin and irradiated CD1 mouse embryonic fibroblasts (MEFs) were plated as a confluent layer of feeder cells. mES cells were seeded at a density of 50,000 cells per well of a six-well plate and were split every 3–4 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from 500,000 - 1 million mES cells using phenol-chloroform-isoamyl alcohol (Thermo) extraction. 500ng of purified DNA was subjected to sonication on a Covaris S220 sonicator (10% duty cycle, Intensity 5, 200 cycles/burst, 6 one minute cycles).
After sonication fragmented DNA was end repaired and A-tailed using Ultra II End Repair/dA-Tailing Module (NEB) then adapters (Broad Institute, single index P7) were ligated using Blunt/TA Ligase Master Mix (NEB). Both protocols were carried out as recommended by the manufacturer. MagMeDIP kit (Diagenode) was used for precipitation and wash steps according to manufacturer’s recommendations. Libraries were PCR amplified with universal primers to add the P7/P5 graft sites using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) then size selected between 200-700bp on an E-Gel Agarose Gels 2% (Thermo). DNA was purified using MinElute Gel Extraction Kit (Qiagen). Purified DNA was quantified using Bioanalyzer HS DNA (Agilent) and qBit HS dsDNA (Thermo) kits then sequenced on a HiSeq 4000 system.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
MeDIP-seq
|
| Data processing |
The MeDIP sequencing data were aligned to the mouse reference genome mm9 using segemehl (v0.2.0, default parameters) and the alignment was filtered to remove unusual insert sizes (>1kb) and multiple mappers.
Post-processing was done using the R package QSEA with the DKOL methylation rates as reference to obtain position-wise read counts.
Genome build: mm9
processed data files format and content: bigWig coverage files
|
| |
|
| Submission date |
Sep 23, 2020 |
| Last update date |
Jun 18, 2021 |
| Contact name |
Helene Kretzmer |
| E-mail(s) |
kretzmer@molgen.mpg.de
|
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Genome Regulation
|
| Lab |
Meissner Lab
|
| Street address |
Ihnestraße 63
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE158460 |
Dnmt1 has global de novo methylation activity and is specifically targeted to transposable elements |
|
| Relations |
| BioSample |
SAMN16249144 |
| SRA |
SRX9179182 |
