1. 染色质构象捕获实验技术探讨 李西川 PhD 2015-11-11

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

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

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

5. Looping Model
Linking Model

6. 3C: Chromasome Conformation Capture
MCB –

7. Primer Design

8. An Example of 3C Assay

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

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

11. Find Cis-elements by 3C
Cancer Cell 25, 575–589, May 12, 2014

12. 3C Protocol (1) harvest cells. (2) wash the cells with PBS once.
(3) cross-linking the cells with 2% Formaldehyde (10ml RPMI ml 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

14. 3C-based Technologies

15. 4C Circular Chromasome Conformation Capture & Chromasome Conformation Capture on Chip

16. Circular Chromasome Conformation Capture

17. e4C

18. e4C Protocol
Nat Protoc Jun 21;7(7):

19. Examples of 4C Assay Cell Stem Cell. 2013 Jul 3;13(1):36-47
Nature Mar 20;507(7492):371-5.

20. Hi-C
Hi-C实验原理

21. Thank you!
李西川：