Epigenetic comparison of human DNA in specific human tissues to human DNA in transgenic mouse tissues
Epigenetic comparison of human DNA in specific human tissues to human DNA in transgenic mouse tissues
doi:10.1016/j.ymeth.2009.03.001; Crosslinked animal livers were dounce homogenized into a single cell suspension and rinsed with ice cold PBS. Each pellet of cross-linked material was resuspended in 10 ml of LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 C. Pellets were then rinsed in 10 ml of LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA) and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 C. The nuclei preparation was resuspended in  3 ml LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine). Chromatin in the nuclei fraction was fragmented to an average length of 300 bp by sonication.  After sonication 300  l of 10% Triton X-100 was added to the 3 ml of sonicated lysate and debris was removed by centrifugation  at 20,000 x rcf for 10 minutes at 4 C.
Experimental Factor: organism
Mus musculus
Experimental Factor: genotype
wild type
Experimental Factor: immunoprecipitate
H3K9me3
Experimental Factor: organism part
liver
Experimental Factor: sex
male