A native chromatin extraction method based on salicylic acid coated magnetic nanoparticles and characterization of chromatin

Abstract

Native chromatin contains valuable genetic, epigenetic and structural information. Though DNA and nucleosome structures are well defined, less is known about the higher-order chromatin structure. Traditional chromatin extraction methods involve fixation, fragmentation and centrifugation, which might distort the higher-order structural information of native chromatin. We present a simple approach to isolate native chromatin from cultured mammalian cells using salicylic acid coated magnetic nanoparticles (SAMNPs). Chromatin is magnetically separated from cell lysates without any filtration or high-speed centrifugation. The purified chromatin is suitable for the examination of histone modifications and other chromatin associated proteins as confirmed by western blotting analysis. The structure of chromatin was determined by confocal fluorescence microscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM). High-resolution AFM and TEM images clearly show a classical bead-on-a-string structure. The higher-order chromatin structure is also determined via electron microscopy. Our method provides a simple, inexpensive and an environmentally friendly means to extract native chromatin not possible before, suitable for both biochemical and structural analysis.

Chromatin hierarchical branching visualized at the nanoscale by electron microscopy

“…Please do not adjust margins indicate that chromatin has fibers with various thicknesses and holds a branching structure, 25 consistent with this study.…”

Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin

“…And due to short half lives of RNA, CARs degrade during the prolonged extraction process causing a bias for the full sequence analysis of CARs. In contrast, chromatin isolation by the established magnetic isolation process is fast and convenient [6], although there is still the issue of free RNA adsorbing to the magnetic nanoparticle surface, which could introduce artifacts for down-stream sequence analysis of CARs. Thus, it is essential to remove free RNA before transcriptomic analysis of CARs.…”

“…In a compared study, 10 μg of chromatin was extracted from 1 million MCF7 cells using our previously reported method [6]; while 14 μg of chromatin was extracted from the same amount of MCF7 cells using the magnetophoretic chromatography method. The extracted chromatin was visualized by TEM, as shown in Fig.…”

“…The assay could be applied to extract CARs to other cells such as Hela cells [18], non-neoplastic S1 HMT-3522 human mammary epithelial cells [6] as well as human blood white cells [7]. The total assay time including cell separation, chromatin collection and chromatin associated RNA extraction was less than 2h.…”

“…In contrast to the conventional method that requires high speed centrifugation for a prolonged period of time to purify soluble chromatin, in our previous studies, we developed methods using magnetic nanoparticles, for example salicylic acid coated magnetic nanoparticles (SAMNPs), for a rapid, unbiased and cost-effective method for the purification of chromatin [6, 7]. When free RNA (most of these are rRNAs and mRNAs) and chromatin were released from lysed mammalian cells, these nucleic acids were non-specifically bound to SAMNPs forming free RNA-SAMNPs, chromatin-SAMNPs complexes.…”