Dilworth et al. [ 32] report that NUP2 (part of the nuclear pore complex) localizes in the nucleus with yeast telomeres; ChIP-chip experiments reveal that NUP2 has a telomere binding preference.
In order to investigate the significance of histone modification changes in specific regions of Sox2, Oct4 and β-globin in transplanted nuclei, we have conducted ChIP analysis.
Finally, nuclei were lyzed in ChIP nuclear lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA (pH 8.0), 1% SDS, and protease inhibitors) and incubated 5 min on ice.
For the E(Z) ChIPs, nuclei were fixed for 20 min instead of the standard 15 min.
For native ChIPs nuclei were extracted and isolated over a sucrose cushion, resuspended in MNase buffer (0.32 M sucrose, 50 mM Tris-HCl pH 7.5, 4 mM MgCl2, 1 mM CaCl2) and digested with 10 U MNase (Thermo Fisher Scientific Inc., Waltham, MA) for at least 15 min at 37°C, so that mononuceosomes were released.
The yield of sorted nuclei in sort buffer (PBS) was determined with a hemacytometer, and 50 200 K nuclei were used for each ChIP.
To validate H3K9me3 at frq, we also performed the H3K9me3 ChIP on isolated nuclei and found H3K9me3 levels above Δdim-5.
To support the sequence-specific binding by HIF-1 to HRE-1 during hypoxic conditions within the nucleus of live cells we preformed ChIP assays.
One way to reduce costs is to genotype nucleus herd selection candidates using a low-density chip and to use high-density chip genotyping for animals that are used as parents in the nucleus breeding herd.
Regarding ChIP assays, purification of nuclei, preparation of chromatin by micrococcal nuclease digestion (to obtain fragments of predominantly one to five nucleosomes) and immunoprecipitation with anti-PC3/Tis21 antibody, were performed as described previously, with minor modifications [37].
