Abstract
Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors’ binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform μChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.
Keywords: Chromatin, PCR, MicroChIP, Limited, Astrocyte
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. μChIP of the histone mark H3K9Acetyl with increasing amount of antibody in mouse cortical astrocytes. Real-time PCR was performed in the promoter region of Pax6 (F: cggtgaaagaagccactagg, R: agggaacacaccaactttcg) and in the promoter region of T-cell receptor (TCR) (F: cttacaccccaaacctccaa, R: gggaggatgaggagaaaagg). Figure 2. μChIP of overexpressed HA-tagged Neurogenin2 in NS-5 cells with increasing amount of antibody. Real-time PCR was performed with primers recognizing the promoter region of the Neurogenin2 (Neurog2) target gene Fbxw7 (F: gcgctttgaagaaagacctg, R: gttttcaaggggcgaatgta) and the ORF region of Dll1, not bound by Neurog2 (F: gtctcaggaccttcacagtag, R: gagcaaccttctccgtagtag). Figure 3. Optimal chromatin solution after sonication of 500,000 astrocytes in 500 μl lysis buffer for 10 min (30 sec on/30 sec off) setting H, Diagenode Bioruptor. Two samples (10 μl each) were run on a 1% agarose gel alongside a DNA ladder.
Recipes
Note: Prepare all the solutions fresh from stock solutions before starting and keep on ice.
Acknowledgments
This work was supported by a project grant from the Wellcome Trust (WT091800MA) and a Grant-in-Aid from the Medical Research Council (U117570528) to F. G. This protocol was established with methods from Peggy Farnham's laboratory and with methods published by François Guillemot's laboratory. We are grateful to Dr Koji Oishi for critical reading of the manuscript.
References
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