title: NextSeq 500 paired end sequencing; DNA- and RNA-seq to study the impact of 5-formylcytosine on nucleosome organization and gene expression in embryonic mouse tissues and stem cells
design: DNA- and RNA-seq to study the impact of 5-formylcytosine on nucleosome organization and gene expression in embryonic mouse tissues and stem cells
library_name: brain_reconst_wt2_p
library_construction_protocol: 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: Experimental Factor: protocol
attributes.value: Micrococcal Nuclease digestion
attributes.tag: Experimental Factor: organism
attributes.value: Mus musculus
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype
attributes.tag: Experimental Factor: organism part
attributes.value: hindbrain
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: none