西亚试剂： H3K4me3 epigenomic landscape derived from ChIP-Seq of 1 00

Epigenetic regulation is crucial to the establishment and maintenance of the identity of a cell. Recent studies suggest that transcription is implemented amongst a mixture of various histone modifications1. It has also been recognized that to interrogate function of genetic information, comprehensively systematic profiling of the epigenome in multiple cell stages and types is required2. Chromatin immunoprecipitation (ChIP) has become one of the most critical assays to investigate the complex DNA-protein interactions3. Combined with profiling technologies such as microarrays (ChIP-on-chip) or high-throughput sequencing (ChIP-Seq), this assay becomes a great tool to study the epigenetic regulatory networks in cells4,5,6. However, the ChIP process produces limited amount of DNA due to the low yield of antibody pull-down, DNA damage during fragmentation and cleavage of DNA-protein complex, and complicated downstream analysis7. The conventional approaches have to consume a considerable amount of samples, typically 106 - 107 cells, to overcome this low-yield issue and obtain reliable results5. This limitation also restricts ChIP applications from precious primary tissue samples such as early embryonic cells or rare tumor stem cells.

ChIP-Seq, compared with ChIP-on-Chip, deeply sequences the target DNA fragments and generates highly comprehensive data with higher resolution, fewer artifacts, greater coverage and larger dynamic range6. Although recent application of automated microfluidic ChIP (AutoChIP) was successfully performed using 2 000 cells through locus-specific analysis by qPCR8, such assays do not achieve the comprehensiveness afforded by DNA sequencing approaches. Recently, several approaches have been developed to perform ChIP-Seq using as low as 10 000 or even only 5 000 cells7,9,10,11,14. However, all of these methods rely on ChIP reactions in tens of microliters and preamplification of ChIP product before sequencing library preparation, either through linear amplification (by in vitro transcription) or exponential amplification (by PCR), both of which potentially introduce significant bias. Adli et al.7 reported a modified protocol to realize the ChIP-Seq using 10 000 cells by revising the random primers used in amplification to reduce the primer self-annealing, with an optimized PCR condition to cover the GC-rich regions. Ng et al.14 developed another protocol to perform ChIP-Seq of H3K4me3 modification using 10 000 mouse primordial germ cells, requiring pre-amplification before the sequencing library preparation. Sachs et al.15 reported a chromatin immunoprecipitation study with low number of cells without pre-amplification, however, it needs at least 50 000 cells as starting material. Here we present a new method that implements a microfluidic device to facilitate the ChIP process, providing a technology to obtain the high-quality ChIP-Seq data from merely 1 000 mammalian cells, with no need of pre-amplification. The whole ChIP process has been greatly shortened to 8 h. Through this method, we have accomplished, for the first time, a rapid, semi-automated, and highly sensitive ChIP assay to investigate the genome-wide landscape of histone modification H3K4me3 using 1 000 mouse epiblast cells at E6.5, and found that the H3K4me3 landscape of post-implantation epiblast is more similar to that of the mEpiSCs than that of mESCs.

Microfluidic devices are the ideal reaction systems for handling small number of cells. We fabricated our PDMS-based device to treat four samples in parallel on a single chip (Figure 1A). Each reaction pipeline could accept no more than 1 200 cells. We first performed the whole ChIP assay on a microfluidic device, including trapping the magnetic beads that pre-incubated with antibody, binding of the sonicated chromatin fragments with H3K4me3 to the beads, and washing away the chromatin fragments without H3K4me3 modification. Alternatively, the micro-device can be used for fragmentation of chromatins through micrococcal nuclease (MNase) treatment on chip, after the cell permeabilization. After the enrichment we collected the DNA out of the device and finished the sequencing library construction in microcentrifuge tubes. We eliminated preamplification of the ChIP product. Instead, we combined the end-repair, adenylation, and ligation steps in a one-tube reaction and then used carrier DNA to facilitate high-efficiency purification of the ligated DNA product. Then we used PCR to amplify these ligated DNAs to get nanogram amount of DNA for sequencing. Conventionally, the experimental process is tedious, taking at least two days to complete the sample treatment3. However, our new protocol can greatly accelerate the whole process; the complete experimental procedure, including microfluidic-based cell permeabilization, chromatin fragmentation, antibody pull-down, and many washing steps, can be completed within 8 h.

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