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Merck

In situ Chromatin Interaction Analysis Using Paired-End Tag Sequencing.

Current protocols (2021-08-06)
Ping Wang, Yuliang Feng, Kun Zhu, Haoxi Chai, Ya-Ting Chang, Xiaofei Yang, Xiyuan Liu, Chen Shen, Eva Gega, Byoungkoo Lee, Minji Kim, Xiaoan Ruan, Yijun Ruan
RESUMEN

Chromatin Interaction Analysis Using Paired-End Tag Sequencing (ChIA-PET) is an established method to map protein-mediated chromatin interactions. A limitation, however, is that it requires a hundred million cells per experiment, which hampers its broad application in biomedical research, particularly in studies in which it is impractical to obtain a large number of cells from rare samples. To reduce the required input cell number while retaining high data quality, we developed an in situ ChIA-PET protocol, which requires as few as 1 million cells. Here, we describe detailed step-by-step procedures for performing in situ ChIA-PET from cultured cells, including both an experimental protocol for sample preparation and data generation and a computational protocol for data processing and visualization using the ChIA-PIPE pipeline. As the protocol significantly simplifies the experimental procedure, reduces ligation noise, and decreases the required input of cells compared to previous versions of ChIA-PET protocols, it can be applied to generate high-resolution chromatin contact maps mediated by various protein factors for a wide range of human and mouse primary cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and data generation Support Protocol: Bridge linker preparation Basic Protocol 2: Data processing and visualization.

MATERIALES
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Roche
cOmplete, Mini, conjunto de inhibidores de proteasas sin EDTA, Protease Inhibitor Cocktail Tablets provided in a glass vial, Tablets provided in a glass vial
Sigma-Aldrich
Glicina, ReagentPlus®, ≥99% (HPLC)
Sigma-Aldrich
TWEEN® 20, for molecular biology, viscous liquid
Millipore
Triton X-100, Hydrogenated, PROTEIN GRADE® Detergent, 10% Solution, Sterile-Filtered, Non-ionic detergent used for the solubilization of membrane-bound proteins.