An optimized protocol for isolating primary epithelial cell chromatin for ChIP.

ABSTRACT: A critical part of generating robust chromatin immunoprecipitation (ChIP) data is the optimization of chromatin purification and size selection. This is particularly important when ChIP is combined with next-generation sequencing (ChIP-seq) to identify targets of DNA-binding proteins, genome-wide. Current protocols refined by the ENCODE consortium generally use a two-step cell lysis procedure that is applicable to a wide variety of cell types. However, the isolation and size selection of chromatin from primary human epithelial cells may often be particularly challenging. These cells tend to form sheets of formaldehyde cross-linked material in which cells are resistant to membrane lysis, nuclei are not released and subsequent sonication produces extensive high molecular weight contamination. Here we describe an optimized protocol to prepare high quality ChIP-grade chromatin from primary human bronchial epithelial cells. The ENCODE protocol was used as a starting point to which we added the following key steps to separate the sheets of formaldehyde-fixed cells prior to lysis. (1) Incubation of the formaldehyde-fixed adherent cells in Trypsin-EDTA (0.25% room temperature) for no longer than 5 min. (2) Equilibration of the fixed cells in detergent-free lysis buffers prior to each lysis step. (3) The addition of 0.5% Triton X-100 to the complete cell membrane lysis buffer. (4) Passing the cell suspension (in complete cell membrane lysis buffer) through a 25-gauge needle followed by continuous agitation on ice for 35 min. Each step of the modified protocol was documented by light microscopy using the Methyl Green-Pyronin dual dye, which stains cytoplasm red (Pyronin) and the nuclei grey-blue (Methyl green). This modified method is reproducibly effective at producing high quality sheared chromatin for ChIP and is equally applicable to other epithelial cell types.

SUBMITTER: Browne JA 

PROVIDER: S-EPMC4074041 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature