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MOWChIP-seq for low-input and high-throughput profiling of genome-wide histone modifications

ABSTRACT: Epigenetic mechanisms such as histone modifications and DNA methylation form a critical layer of control that regulates gene activities. Histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling various histone modifications at the genome scale using primary tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a particular cell type, isolation of a cell type of interest from primary tissues is often necessary to generate a homogeneous cell population and such operation tends to yield a low number of cells. In addition, high-throughput processing is desired in such effort due to the number of histone marks of interests and the potential volume of samples in a hospital setting. In this protocol, we describe detailed information on device fabrication, setup, and operation of microfluidic oscillatory washing-based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling histone modifications using as few as 30-100 cells per assay with a throughput as high as 8 assays in a run. The critical step of MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads followed by a vigorous microfluidic oscillatory washing. The ChIP process is semi-automated for reduced labor and improved reproducibility. We have used the protocol to study a number of histone modifications in various types of mouse and human tissue types ranging from isolated nuclei of brain cells to cell subtypes isolated from human breast tissues.

ORGANISM(S): Mus musculus

PROVIDER: GSE123606 | GEO | 2019/08/31

REPOSITORIES: GEO

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Project description:Post-hybridization washing is an essential part of microarray experiments. Both, the quality of the experimental washing protocol and the adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities. We conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of washing cycles. Particularly, three Affymetrix GeneChip HGU133plus2 arrays were hybridized and equilibrated for 16 hours in the hybridization oven. For one of the three arrays washing and staining was performed according to the manufacturer’s instructions. For another array the first scan was done immediately after low stringent wash and staining without intermitting stringent washing. Then, the array was stringently washed and scanned in alternating order three more times where each washing step consists of a definite number of washing cycles. The third array was low stringently washed followed by two stringent washing cycles and staining before the first scan. Subsequently it was analogously processed as array A. All three chips are repeatedly processed in a second series of alternating wash/scan-cycles which was performed using the same protocol for each chip as in the first series as described above. As in the first series the arrays were also stained a second time to compensate for any loss of bleached fluorescent dye. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. The washing function allows calibrating probe intensities for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures especially in the limit of small and large values.