Chromatin interactions functionally affect genome architecture and gene regulation,but to date,only fresh samples must be used in High-through chromosome conformation capture(Hi-C)to keep natural chromatin conformatio...Chromatin interactions functionally affect genome architecture and gene regulation,but to date,only fresh samples must be used in High-through chromosome conformation capture(Hi-C)to keep natural chromatin conformation intact.This requirement has impeded the advancement of 3 D genome research by limiting sample collection and storage options for researchers and severely limiting the number of samples that can be processed in a short time.Here,we develop a freeze substitution Hi-C(FS-Hi-C)technique that overcomes the need for fresh samples.FS-Hi-C can be used with samples stored in liquid nitrogen(LN2):the water in a vitreous form in the sample cells is replaced with ethanol via automated freeze substitution.After confirming that the FS step preserves the natural chromosome conformation during sample thawing,we tested the performance of FS-Hi-C with Drosophila melanogaster and Gossypium hirsutum.Beyond allowing the use of frozen samples and confirming that FS-Hi-C delivers robust data for generating contact heat maps and delineating A/B compartments and topologically associating domains,we found that FS-HiC outperforms the in situ Hi-C in terms of library quality,reproducibility,and valid interactions.Thus,FS-HiC will probably extend the application of 3D genome structure analysis to the vast number of experimental contexts in biological and medical research for which Hi-C methods have been unfeasible to date.展开更多
基金supported by funding from the National Natural Science Foundation of China,China(grants 31621005 and 31600093 to F.G.L.)the Young Elite Scientists Sponsorship Program by CAST(2019zx08010033-002-012 to Z.E.Y.)Central Public-interest Scientific Institution Basal Research Fund(Y2020PT13)。
文摘Chromatin interactions functionally affect genome architecture and gene regulation,but to date,only fresh samples must be used in High-through chromosome conformation capture(Hi-C)to keep natural chromatin conformation intact.This requirement has impeded the advancement of 3 D genome research by limiting sample collection and storage options for researchers and severely limiting the number of samples that can be processed in a short time.Here,we develop a freeze substitution Hi-C(FS-Hi-C)technique that overcomes the need for fresh samples.FS-Hi-C can be used with samples stored in liquid nitrogen(LN2):the water in a vitreous form in the sample cells is replaced with ethanol via automated freeze substitution.After confirming that the FS step preserves the natural chromosome conformation during sample thawing,we tested the performance of FS-Hi-C with Drosophila melanogaster and Gossypium hirsutum.Beyond allowing the use of frozen samples and confirming that FS-Hi-C delivers robust data for generating contact heat maps and delineating A/B compartments and topologically associating domains,we found that FS-HiC outperforms the in situ Hi-C in terms of library quality,reproducibility,and valid interactions.Thus,FS-HiC will probably extend the application of 3D genome structure analysis to the vast number of experimental contexts in biological and medical research for which Hi-C methods have been unfeasible to date.
