染色质构象捕获实验技术探讨.ppt

染色质构象捕获实验技术探讨 李西川 PhD 2015-11-11,Chromosome,DNA,High-Order Chromatin Organization,And Epigenetical Regulation,Neurogastroenterol Motil. 2012 Dec; 24(12): 10541068,Spatial organization of chromosome,Annu Rev Genomics Hum Genet. 2013;14:67-84,Neurogastroenterol Motil. 2012 Dec; 24(12): 10541068,Chromasome Conformation,A schematic rendering of a 2.1 Mb region on chromosome 20 (48.7850.88 Mb),Cell. 2014 Dec 18;159(7):1665-80,Chromatin interactions within and between chromosomes,Methods. 2012 Nov; 58(3): 10.1016,Looping Model,Linking Model,3C: Chromasome Conformation Capture,MCB.25.8.32203231.2005,Primer Design,An Example of 3C Assay,Cancer Cell 25, 575589, May 12, 2014,Chromosomal Loops in SHC1 Gene Cluster,Example II of 3C Assay,Curr Opin Cell Biol. 2014 Feb; 0: 6978.,Find Cis-elements by 3C,Cancer Cell 25, 575589, May 12, 2014,3C Protocol,(1) harvest cells. (2) wash the cells with PBS once. (3) cross-linking the cells with 2% Formaldehyde (10ml RPMI1640+0.54ml formaldehyde), 10 min at RT. (4) add 0.5ml 2.5M Glycine, 5min at RT. (5) Harvest cells. Wash the cells with cold PBS once. (6) suspend the cells in 30ml Lysis buffer(10 mM Tris-Cl pH 8.0, 10mM NaCl, 0.2% NP-40, 1mM DTT), on ice for 1hr. (7) harvest the cells and resuspend the cells in the restriction endonuclearse buffer containing 0.3% SDS (500ul/10E7 cells), 37 for 1 hr, shaking. (8) add 9ul Triton/500ul sample, 37 for 1 hr, shaking. (9) add 8ul restriction endonuclearse to 100 ul sample, 37 overnight (10) add 19ul 10% SDS to 100ul sample, 65 20min. (11) 40ul sample to 500ul ligation system. 400ul H2O, 50ul T4 ligase buffer(promega), 40ul DNA sample, 5ul Triton; 37 1hr shaking. (12) add 5ul T4 ligase, 16 4.5hr, RT 30min. (13) add proteinase K to 200ug/ml, 65 overnight. (14) add Rnase, 37 30min. (15) purify DNA (16) PCR,3C-based Technologies,4C Circular Chromasome Conformation Capture & Chromasome Conformation Capture on Chip,Circular Chromasome Conformation Capture,e4C,e4C Protocol,Nat Protoc. 2012 Jun 21;7(7):1335-50.,Examples of 4C Assay,Nature. 2014 Mar 20;