Maria Gridina1, Evgeniy Mozheiko1, Emil Valeev1,2, Ludmila P Nazarenko3, Maria E Lopatkina3, Zhanna G Markova4, Maria I Yablonskaya5, Viktoria Yu Voinova5, Nadezhda V Shilova4, Igor N Lebedev3, Veniamin Fishman6,7. 1. Institute of Cytology and Genetics SB RAS, Lavrentjeva ave 10, Novosibirsk, Russia. 2. Novosibirsk State University, Pirogova str., 2, Novosibirsk, Russia. 3. Research Institute of Medical Genetics, Tomsk National Research Medical Center, Kooperativny Str, 5, Tomsk, Russia. 4. Research Centre for Medical Genetics, Moskvorechie str., 1, Moscow, Russia. 5. Clinical Research Institute of Pediatrics Named After Acad. Y.E. Veltischev, Moscow, Russia. 6. Institute of Cytology and Genetics SB RAS, Lavrentjeva ave 10, Novosibirsk, Russia. minja-f@ya.ru. 7. Novosibirsk State University, Pirogova str., 2, Novosibirsk, Russia. minja-f@ya.ru.
Abstract
BACKGROUND: The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation. RESULTS: In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming. CONCLUSIONS: We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.
BACKGROUND: The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation. RESULTS: In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming. CONCLUSIONS: We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.
Authors: Jay Ghurye; Arang Rhie; Brian P Walenz; Anthony Schmitt; Siddarth Selvaraj; Mihai Pop; Adam M Phillippy; Sergey Koren Journal: PLoS Comput Biol Date: 2019-08-21 Impact factor: 4.475
Authors: Christopher J Troll; Nicholas H Putnam; Paul D Hartley; Brandon Rice; Marco Blanchette; Sameed Siddiqui; Javkhlan-Ochir Ganbat; Martin P Powers; Ramesh Ramakrishnan; Christian A Kunder; Carlos D Bustamante; James L Zehnder; Richard E Green; Helio A Costa Journal: J Mol Diagn Date: 2018-12-31 Impact factor: 5.568
Authors: Wenxiu Ma; Ferhat Ay; Choli Lee; Gunhan Gulsoy; Xinxian Deng; Savannah Cook; Jennifer Hesson; Christopher Cavanaugh; Carol B Ware; Anton Krumm; Jay Shendure; C Anthony Blau; Christine M Disteche; William S Noble; ZhiJun Duan Journal: Methods Date: 2018-01-31 Impact factor: 3.608
Authors: Nariman Battulin; Veniamin S Fishman; Alexander M Mazur; Mikhail Pomaznoy; Anna A Khabarova; Dmitry A Afonnikov; Egor B Prokhortchouk; Oleg L Serov Journal: Genome Biol Date: 2015-04-14 Impact factor: 13.583
Authors: Noelia Díaz; Kai Kruse; Tabea Erdmann; Annette M Staiger; German Ott; Georg Lenz; Juan M Vaquerizas Journal: Nat Commun Date: 2018-11-29 Impact factor: 14.919