TY - JOUR

T1 - A cookbook for DNase Hi-C

AU - Gridina, Maria

AU - Mozheiko, Evgeniy

AU - Valeev, Emil

AU - Nazarenko, Ludmila P.

AU - Lopatkina, Maria E.

AU - Markova, Zhanna G.

AU - Yablonskaya, Maria I.

AU - Voinova, Viktoria Yu

AU - Shilova, Nadezhda V.

AU - Lebedev, Igor N.

AU - Fishman, Veniamin

N1 - Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/3/20

Y1 - 2021/3/20

N2 - 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.

AB - 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.

KW - A549

KW - DNAse I

KW - Genome organization

KW - Hi-C

KW - Human peripheral blood

KW - K562

KW - LNCaP

UR - http://www.scopus.com/inward/record.url?scp=85103169929&partnerID=8YFLogxK

U2 - 10.1186/s13072-021-00389-5

DO - 10.1186/s13072-021-00389-5

M3 - Article

C2 - 33743768

AN - SCOPUS:85103169929

VL - 14

JO - Epigenetics and Chromatin

JF - Epigenetics and Chromatin

SN - 1756-8935

IS - 1

M1 - 15

ER -