An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results

Abstract

Chromatin immunoprecipitation (ChIP) is a commonly used technique to investigate which parts of a genome are bound by a particular protein. The result of ChIP is often interpreted in a binary manner: bound or not bound. Due to this focus, ChIP protocols frequently lack the ability to quantitatively compare samples with each other, for example in a time series or under different growth conditions. Here, using the yeast S. cerevisiae transcription factors Cbf1, Abf1, Reb1, Mcm1 and Sum1, we optimized the five major steps of a commonly used ChIP protocol: cross-linking, quenching, cell lysis, fragmentation and immunoprecipitation. Quenching with glycine is inefficient and can lead to large degrees of variability, an issue that is resolved by using tris(hydroxymethyl)aminomethane (Tris). Another source of variability is degradation of the protein of interest during the procedure. Enzymatic cell lysis with zymolyase can lead to extensive protein degradation, which is greatly reduced by mechanical lysis through bead beating. Degradation also occurs during sonication of chromatin, affecting large proteins in particular. An optimal mix of protease inhibitors and cross-linking with a higher percentage of formaldehyde reduces the extent of this degradation. Finally we also show that the immunoprecipitation step itself can be greatly improved with magnetic beads and optimized incubation/washing steps. The study results in a highly optimized protocol, which is shorter, easier to perform and has a stronger, more reproducible signal with less background. This protocol is presented in detail. In addition, the results highlight the greatest sources of variability in many other protocols, showing which steps are important to focus on for reproducible and quantitatively comparable ChIP experiments.

Genome‐wide off‐rates reveal how DNA binding dynamics shape transcription factor function

“…Having ascertained Abf1 depletion, we next determined whether the system can be used to measure TF mean residence times (defined as 1/ k off ) at different sites across the genome. As published elsewhere (preprint: de Jonge et al , 2019), first the ChIP protocol was extensively optimized at almost all steps, to yield results better comparable between different time points. Next, to determine off‐rates, Abf1 was depleted from the nucleus and its binding levels were measured genome‐wide using the optimized ChIP‐seq protocol at 11 time points during 90 min of depletion, all in biological triplicate (Fig 1A).…”

Genome-wide off-rates reveal how DNA binding dynamics shape transcription factor function

“…A second consideration is that the ChIP or genomic location protocol requires results that are comparable between time points. Here we first extensively optimized almost all ChIP protocol steps to achieve this (de Jonge et al, 2019). Some limitations still remain.…”

“…Having ascertained Abf1 depletion, we next determined whether the system can be used to measure TF residence times (1/k off ) at different sites across the genome. As published elsewhere (de Jonge et al, 2019), first the ChIP protocol was extensively optimized at almost all steps, to yield results better comparable between different timepoints. Next, to determine off-rates, Abf1 was depleted from the nucleus and its binding levels were measured genome-wide using the optimized ChIP-seq protocol at 11 time-points during 90 minutes of depletion, all in biological triplicate (Fig 1A).…”

An Optimized Chromatin Immunoprecipitation Protocol for Quantification of Protein-DNA Interactions

“…This protocol facilitates sensitive and reproducible quantitation of protein-DNA interactions. For complete details on the use and execution of this protocol, please refer to ( de Jonge et al., 2019 ).…”

“…For complete details on the use and execution of this protocol, please refer to ( de Jonge et al., 2019 ).…”