organism: Mus musculus
description: Protocols: TDG KO mice contain a deletion of the coding sequence for the catalytic domain of TDG, which is replaced by a neomycin-resistant cassette. Genotyping was carried out using DNA isolated from the yolk sac as template with the following primers:TDG KO/WT forward: 5′-GGGCAGCAAGGATCTGTCTA-3′TDG KO/WT reverse: 5′-GCCAGTTCCTCTGACACTTAGC-3′Mice were maintained as heterozygotes and intercrossed to generate homozygous knockout embryos for analysis. Tissues were collected from timed matings with the day of the copulation plug as E0.5. Embryos were dissected into hindbrain and heart. Tissues were collected from timed matings with the day of the copulation plug as E0.5. Embryos were dissected into hindbrain and heart. Genomic DNA was extracted from mouse embryonic tissues at 11.5 days using the DNeasy Blood & Tissue kit (Qiagen) For native MNase sequencing, tissues obtained from mouse embryos were homogenized, washed with 5mL cold 1xPBS and pelleted at 300g at 4° C for 10 min. Cell pellet was resuspended in 500 µL of ice-cold NP40 lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 0.15 mM spermine and 0.5 mM spermidine) and incubated on ice for 5 min. Nuclei were subsequently pelleted at 120g at 4° C for 10 min, washed with MNase digestion buffer (10 mM Tris-HCl pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine and 0.5 mM spermidine) and resuspended in MNase digestion buffer containing 1 mM CaCl2. MNase (5 units) were added and the sample was incubated at room temperature for 5 min before the addition of MNase digestion buffer and MNase stop buffer (100 mM EDTA and 10 mM EGTA pH 7.5). Proteinase K and 20% SDS was added and the mixture was incubated at 37° C overnight. After chloroform/ phenol extraction, 2 µL of heat-treated RNase A (10 mg/ mL) was added to each sample and incubated at 37° C for 2 h. After another round of chloroform/ phenol extraction, the digested genomic DNA was obtained by ethanol precipitation. DNA libraries were obtained by end repair, A-tailing and adapter ligation (NEBNext Ultra II DNA library Prep kit) following the manufacturer's guideline.
attributes.tag: ENA first public
attributes.value: 2018-10-22
attributes.tag: ENA last update
attributes.value: 2017-12-07
attributes.tag: External Id
attributes.value: SAMEA104420746
attributes.tag: INSDC center alias
attributes.value: Senior research associate
attributes.tag: INSDC center name
attributes.value: Senior research associate
attributes.tag: INSDC first public
attributes.value: 2018-10-22T17:03:09Z
attributes.tag: INSDC last update
attributes.value: 2017-12-07T13:57:46Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6271:heart_reconst_wt1
attributes.tag: age
attributes.value: embryonic day 11.5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: heart
attributes.tag: sample name
attributes.value: E-MTAB-6271:heart_reconst_wt1
attributes.tag: sex
attributes.value: mixed