Abstract
Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has enabled genome-wide epigenetic profiling of numerous cell lines and tissue types. A major limitation of ChIP-seq, however, is the large number of cells required to generate high-quality data sets, precluding the study of rare cell populations. Here, we present an ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) and sequencing method to generate genome-wide histone mark profiles with high resolution from as few as 10(3) cells. We demonstrate that ULI-NChIP-seq generates high-quality maps of covalent histone marks from 10(3) to 10(6) embryonic stem cells. Subsequently, we show that ULI-NChIP-seq H3K27me3 profiles generated from E13.5 primordial germ cells isolated from single male and female embryos show high similarity to recent data sets generated using 50-180 × more material. Finally, we identify sexually dimorphic H3K27me3 enrichment at specific genic promoters, thereby illustrating the utility of this method for generating high-quality and -complexity libraries from rare cell populations.
Highlights
Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has enabled genome-wide epigenetic profiling of numerous cell lines and tissue types
By intersecting our native’ ChIP (NChIP)-seq data sets with RNA-seq libraries generated from 103 male and female E13.5 primordial germ cells (PGCs), we identified a subset of genes involved in meiosis and transforming growth factor-b receptor signalling that show sex-specific differences in expression and H3K27me[3] enrichment in their promoter regions
To improve the yield of chromatin isolated from small samples, we optimized a dilution-based NChIP-seq procedure that can be adjusted to cell sample size
Summary
Complexity of ULI-NChIP-seq libraries from 103 to 105 cells. To improve the yield of chromatin isolated from small samples, we optimized a dilution-based NChIP-seq procedure that can be adjusted to cell sample size. As for H3K9me[3] libraries, using PreSeq[15] to extrapolate the potential complexity of these libraries indicates that even with the lowest input, all of the H3K27me[3] libraries could be several times the required depth to obtain high-quality profiles (Fig. 1c) To determine whether this method can be used to create profiles for active histone marks, we generated ULI-NChIPseq data for H3K4me[3]. To determine sexspecific H3K27me[3] profiles correlation to sex-specific gene expression, we used ULI-NChIP-seq data sets prepared from PGCs purified from the gonads of single male and female E13.5 embryos[19]. These results reveal that at this stage in PGC development, the polycomb pathway may be engaged more frequently in the male germ line to regulate germ cell-specific genes
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