NextSeq 500 paired end sequencing; Epigenomic changes associated with TGF-beta induced EMT in MDCK cells.
Epigenomic changes associated with TGF-beta induced EMT in MDCK cells.
MDCK cells were maintained in DMEM growth medium supplemented with 10% Foetal Bovine Serum (FBS) For TGF-beta treatment, wildtype MDCK were stimulated in culture by adding 5ng of TGF-beta to the medium. TGF-beta was added with every passage of cells, to maintain stimulus. Nuclei were pelleted by centrifugation for 40 min at 4,000g through an 8-ml sucrose cushion (1.2 M sucrose, 60 mM KCL, 15 mM HEPES, 15 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT, 0.2 mM PMSF and protease inhibitor cocktail, pH 7.6). The supernatant was carefully removed, and pelleted nuclei were resuspended in 1 ml of micrococcal digestion buffer (50 mM Tris-HCl pH 7.6, 3 mM CaCl2) and passed five or six times through a size 29–gauge needle. The nuclear extract was digested for 35 min at 37 °C with micrococcal nuclease (New England Biolabs; 40 gel units per 330 μl of extract) to obtain ~80% mononucleosomes. After centrifugation at 10,000g for 5 min, the supernatant (S1) and the resuspended pellet (S2) were dialyzed against 10 mM Tris-HCl pH 7.6, 1 mM EDTA and 0.5 mM EGTA overnight at 4 °C. The S1 and S2 supernatants were then combined and dialyzed against 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.5 mM EGTA, 4% (v/v) glycerol, divided into aliquots and stored at −80 °C. ChIP-Seq/MNase-Seq libraries were constructed using NEBnext Ultra DNA Library Prep kit for Illumina (New England Biolabs).
Research Fellow Tremethick Lab Department of Genome Sciences The John Curtin School of Medical Research The Australian National University
E-MTAB-5637:MDCK_Input-TGFb-rep2_p
Experimental Factor: input DNA
immunoprecipitate