|
| Status |
Public on Feb 24, 2011 |
| Title |
MEF TDG knockout 3 |
| Sample type |
SRA |
| |
|
| Source name |
Mouse embryonic fibroblast cells
|
| Organism |
Mus musculus |
| Characteristics |
genetic background: 129 C57BL/6 mixed
cell type: embryonic fibroblast cells
genotype: TDG knockout
medip antibody: 5-Methylcytosine
antibody manufacturer: Eurogentec
antibody catalog #: BI-MECY-0100
antibody lot #: Lot:070809
mean insert size: 78.94
insert size standard deviation: 25.53
|
| Treatment protocol |
Before differentiation experiments for neuronal differentiation, ESC were grown in the absence of feeder cells for two passages. For embryoid body formation during neuronal differentiation 4 x 10e6 Tdg+/- or Tdg-/- ESC were plated into nonadherent bacterial dishes (Greiner Bio-one, Germany) in differentiation medium (ECM without LIF and with10% FCS) and grown at 37°C with a medium exchange after two days. After four days,5 μM all-trans retinoic acid (RA) was added and cells were further incubated for four days with a medium exchange after two days. Embryoid bodies were washed twice with 1x PBS and dissociated with freshly prepared trypsin solution (0.05% TPCK-treated trypsin in 0.05% EDTA/1x PBS) at 37°C for 3 min. Dissociated embryoid bodies were resuspended in 10 ml differentiation medium and collected by centrifugation at 700g for 5 min at room temperature (RT). The pellet was resuspended in N2 medium (DMEM:F12 nutrient mixture 1:1, 1x N2 supplement) and the cell suspension filtered through a 40 μm nylon cell strainer (BD, USA). Filtered cells were immediately plated onto poly-L-lysine (PLL) and laminin-coated dishes at a density of 5 × 10e6 cells/60 mm dish or 1.5× 10e7 cells/100 mm dish. The N2 medium was exchanged 2 hours after plating and cells were harvested after 4 hours for extraction of genomic DNA.
|
| Growth protocol |
SV40 immortalized MEF cell lines were cultivated in growth medium (DMEM, 10% FCS, 2 mM L-glutamine) at 37°C in a humidified atmosphere containing 5% CO2. ES cells (ESC) were grown in the presence of feeder cells at 37°C in ES cell medium (ECM: DMEM, 15% heat inactivated FCS, LIF (1,000 U/ml), 1x nonessentialamino acids, 1 mM Na-pyruvate, 2 mM L-glutamine and 90 μM β-mercaptoethanol) in a humidified atmosphere containing 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
5ug of DNA was sonicated giving fragment sizes <500bp.Fragments were end repaired, phosphorylated and A-tailed the fragmented DNA and ligated Illumina multiplex adapters to fragments in accordance with the Illumina Multiplex Sample Preparation protocol. These samples were then subjected to MeDIP as described previously (Weber et al. 2005; PMID: 16007088). The immunoprecipitated (IP) sample was purified using Zymo-5 kit according to the manufacturers instructions.The sample isolated by meDIP then underwent gel electrophoresis and library size selection, prior to PCR amplification using Illumina paired-end PCR primers for 18 cycles. Bands were excised to produce libraries with insert sizes of 100-150 bp and 150-200bp, libraries were quantified using an Agilent Bioanalyzer 2100.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
DNA IP against 5-Methylcytosine
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the mouse (July, 2007) genome using the software 'bwa' (http://bio-bwa.sourceforge.net/). Paired reads with a bwa alignment score of <10 were removed from downstream analysis.
|
| |
|
| Submission date |
Feb 23, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Gareth Wilson |
| E-mail(s) |
gareth.wilson@ucl.ac.uk
|
| Organization name |
UCL Cancer Institute
|
| Street address |
23 Huntley Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE27468 |
Methylome analysis of 18 wild-type and mutant mouse ES, NP and MEF cells |
|
| Relations |
| SRA |
SRX046093 |
| BioSample |
SAMN00216770 |
