NextSeq 500 paired end sequencing; Exploring the relationship between 5fC and nucleosomal organization in tissues obtained from mouse embryos E11.5 and synthetic DNA
Exploring the relationship between 5fC and nucleosomal organization in tissues obtained from mouse embryos E11.5 and synthetic DNA
TDG KO mice generation protocol: The Tdg-targeting construct (Supplementary Fig. 1) was generated by replacement of a NarI–PacI fragment enclosing exons 6 and 7 by a neomycin resistance cassette in a cloned fragment spanning exons 5–10 of the Tdg locus. This construct was used to target the Tdg allele in 129 mouse ESCs, which were then used to generate chimaeras and, ultimately, Tdg+/− heterozygotes by backcrossing to C57BL/6. Text taken from: doi:10.1038/nature09672 Breeding and tissue collection: 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, heart, liver and carcass.Text taken from: https://doi.org/10.1186/s13059-016-1001-5. NaBH4 was used to reduce 5-fC to 5-hmC as a control in the nucleosome reconstitution experiment (SLX-11454* samples) to demonstrate that MNase signal is derived from 5-fC. More details can be found in our previous work<http://www.nature.com/nchem/journal/v6/n5/full/nchem.1893.html>. NaBH3CN is a reducing agent which irreversibly traps out the Schiff base interactions (covalent bonds) between 5-fC and histone proteins. It was used in the polymerase stop assays (Polstop* samples). The concentration of NaBH3CN used was 100mM, the concentration where we had observed DNA-histone subunit cross-linking by gel electrophoresis. Genomic DNA and RNA was extracted from mouse embryonic tissues using the DNeasy Blood & Tissue kit (Qiagen). DNA library preparation for sequencing was done using the NEBNext DNA library preparation kit (New England Biolabs Inc.)
Experimental Factor: compound
NaBH4
Experimental Factor: organism part
hindbrain
Experimental Factor: genotype
wild type genotype
Experimental Factor: phenotype
wild type phenotype