This Title All WIREs
How to cite this WIREs title:
WIREs Syst Biol Med
Impact Factor: 4.192

Going low to reach high: Small‐scale ChIP‐seq maps new terrain

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Abstract Chromatin immunoprecipitation (ChIP) enables mapping of specific histone modifications or chromatin‐associated factors in the genome and represents a powerful tool in the study of chromatin and genome regulation. Importantly, recent technological advances that couple ChIP with whole‐genome high‐throughput sequencing (ChIP‐seq) now allow the mapping of chromatin factors throughout the genome. However, the requirement for large amounts of ChIP‐seq input material has long made it challenging to assess chromatin profiles of cell types only available in limited numbers. For many cell types, it is not feasible to reach high numbers when collecting them as homogeneous cell populations in vivo. Nonetheless, it is an advantage to work with pure cell populations to reach robust biological conclusions. Here, we review (a) how ChIP protocols have been scaled down for use with as little as a few hundred cells; (b) which considerations to be aware of when preparing small‐scale ChIP‐seq and analyzing data; and (c) the potential of small‐scale ChIP‐seq datasets for elucidating chromatin dynamics in various biological systems, including some examples such as oocyte maturation and preimplantation embryo development. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates
Native chromatin immunoprecipitation (ChIP) (NChIP) and cross‐linking ChIP (XChIP). In NChIP, the cells are not subjected to formaldehyde fixation, as is the case in XChIP protocols. The DNA of lysed cells may be fragmented by either enzymatic digestion or using sound waves (sonication). After chromatin release, the insoluble fraction is removed by centrifugation. The soluble fraction, containing the released chromatin, is used in immunoprecipitation (IP). Antibody‐coated beads are mixed with the chromatin, and epitopes are allowed to bind. After washing away unspecifically bound material, the chromatin is eluted from the beads. In XChIP, a decross‐linking step is required. Next, DNA is purified, and used to prepare sequencing libraries. Library preparation includes repair of damaged DNA ends, ligation of sequencing adapters to each end of the DNA fragments, and amplification of the ChIP DNA library by polymerase chain reaction (PCR)
[ Normal View | Magnified View ]
Comparison of low cell number cross‐linking ChIP (XChIP), native ChIP (NChIP) and ultra‐low‐input CUT&RUN (uliCUT&RUN). (a) Genome browser tracks show H3K4me3 data from different numbers of ES cells, generated from cross‐linking (X)ChIP (Reanalysis of data from Dahl et al. (), along with a 200‐cell ChIP‐seq experiment using our most recent and optimized protocol [indicated with *, Manaf et al., manuscript in preparation]), native (N)ChIP (Reanalysis of data from Zhang et al. ()), and uliCUT&RUN (Reanalysis of data from Hainer et al. ()). Values on Y‐axes are fragments per kilobasepair per million (FPKM) reads for X‐ChIP and N‐ChIP data, and for Cut&Run, the values represent those of the authors' exported output. Figures were generated using EaSeq (http://easeq.net). (b) Plots of average H3K4me3 signal at transcription start sites from the same datasets as presented in (a). Y‐axes values are as in (a). For comparison between methodologies, note the scale on the y‐axes. In conditions where replicates existed, each line represents the average of two replicates. (c) Two‐dimensional (2D)‐histograms showing the correlation of N‐ChIP and X‐ChIP datasets from two replicates of 5 k and 10 k cells, respectively. Signal was quantified within +/−500 bp of all transcription start sites and values on the X‐axes and Y‐axes are FPKM. r‐values show Pearsson's correlation coefficients
[ Normal View | Magnified View ]
Low complexity and high complexity chromatin immunoprecipitation (ChIP)‐seq libraries. (a) Deeper sequencing of a nearly saturated low complexity library will mostly result in a pile up of reads mapped to the very same genomic coordinates. The practice of filtering for unique reads ensures that such piles of reads that represent the same ChIP DNA fragment will not bias the downstream analysis. (b) Low complexity libraries are recognized by a low number of unique reads due to a low number of ChIP DNA fragments present in the final library. A low complexity library reaches saturation with a relatively low sequencing depth as compared to a high complexity library. Deeper sequencing of a nearly saturated low complexity library will generate only a limited amount of additional unique reads
[ Normal View | Magnified View ]

Browse by Topic

Biological Mechanisms > Cell Fates
Developmental Biology > Developmental Processes in Health and Disease
Laboratory Methods and Technologies > Genetic/Genomic Methods

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts