ChIP-Seq Assays from Mammalian Cartilage and Chondrocytes.

Abstract

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful tool to identify binding profiles of transcriptional regulators and chromatin regulators as well as histone modification patterns in a genome-wide manner. ChIP-seq consists of five major steps: (1) preparation of cells and chromatin, (2) ChIP, (3) ChIP-seq library construction, (4) sequencing of ChIP DNA with a next-generation sequencer (NGS), and (5) computational analysis of sequence data. Recent ChIP-seq studies in skeletal tissues enable us to understand the modes of action of key skeletal regulators, functional interaction among the enhancers bound by the regulators, the complex nature of regulatory inputs, and thereby the gene regulatory landscape in skeletal development. Here we describe a ChIP-seq protocol that we have employed in our studies, with particular focus on chromatin preparation and subsequent ChIP in skeletal cells, including chondrocytes.