染色质构象捕获实验技术探讨 李西川 PhD 2015-11-11

Chromosome DNA And Epigenetical Regulation High-Order Chromatin Organization And Epigenetical Regulation DNA Neurogastroenterol Motil. 2012 Dec; 24(12): 1054–1068

Spatial organization of chromosome Neurogastroenterol Motil. 2012 Dec; 24(12): 1054–1068 Annu Rev Genomics Hum Genet. 2013;14:67-84

Chromasome Conformation Methods. 2012 Nov; 58(3): 10.1016 Chromatin interactions within and between chromosomes A schematic rendering of a 2.1 Mb region on chromosome 20 (48.78–50.88 Mb) Cell. 2014 Dec 18;159(7):1665-80

Looping Model Linking Model

3C: Chromasome Conformation Capture MCB.25.8.3220–3231.2005

Primer Design

An Example of 3C Assay

Chromosomal Loops in SHC1 Gene Cluster Cancer Cell 25, 575–589, May 12, 2014

Example II of 3C Assay Curr Opin Cell Biol. 2014 Feb; 0: 69–78.

Find Cis-elements by 3C Cancer Cell 25, 575–589, 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 Cell Stem Cell. 2013 Jul 3;13(1):36-47 Nature. 2014 Mar 20;507(7492):371-5.

Hi-C Hi-C实验原理

Thank you! 李西川：13512213661 xichuanli@tmu.edu.cn