title: NextSeq 500 paired end sequencing; Epigenomic changes associated with TGF-beta induced EMT in MDCK cells.
design: Epigenomic changes associated with TGF-beta induced EMT in MDCK cells.
library_construction_protocol: MDCK cells were maintained in DMEM growth medium supplemented with 10% Foetal Bovine Serum (FBS) 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. For ChIP, 5 ug of anti- H2A.Z antibodies were used for 150 uq of MNAse digested chromatin.  Samples were then RNAse treated for 30 mins at 37 degrees followed by Proteinase K at 65 degrees for 5 hours to decrosslink and release DNA. Finally, AMPure beads were used to purify the ChIP/Input DNA. ChIP-Seq/MNase-Seq libraries were constructed using NEBnext Ultra DNA Library Prep kit for Illumina (New England Biolabs).
identifiers.namespace: Research Fellow Tremethick Lab Department of Genome Sciences The John Curtin School of Medical Research The Australian National University
identifiers.id: E-MTAB-5637:MDCK_H2AZ-WT-rep2_p