Single-tube linear DNA amplification for genome-wide studies using a few thousand cells

Abstract

Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled to massive parallel sequencing (ChIP-seq) has been achieved for transcription factors and epigenetic modification of chromatin histones with 1,000 to 5,000 cells. LinDA largely simplifies reChIP-seq experiments to monitor co-binding at chromatin target sites. The single-tube design of LinDA is ideal for handling ultrasmall amounts of DNA (<30 pg) and is compatible with automation. The actual hands-on working time is less than 6 h with one overnight reaction. The present protocol describes all materials and critical steps, and provides examples and controls for LinDA. Applications of LinDA for genome-wide analyses of biobank samples and for the study of chromatin conformation and nuclear architecture are in progress.

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Figure 1: Schematic illustration of the LinDA protocol and its key features.
Figure 2: Comparative statistics illustrating peak detection relative to peak size for LinDA–ChIP-seq with different amounts of cells relative to the gold standard.
Figure 3: Comparison of GC content across unamplified versus LinDA-amplified ER-α ChIP profiles.

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Acknowledgements

W.v.G. is supported by the Fondation pour la Recherche Médicale (aide aux projets innovants). This work was supported by funds from the Ligue National Contre le Cancer (laboratoire labelisé), the Institut National du Cancer (INCa) and the European Community contracts LSHC-CT-2005-518417 'EPITRON', LSHG-CT2005-018882 'X-TRA-NET', LSHM-CT2005-018652 'CRESCENDO' and HEALTH-F4-2009-221952 'ATLAS'.

Author information

P.S., L.M.T. and H.G. designed and optimized LinDA. M.-A.M.-P. performed the F9 experiments and did the bioinformatics analysis together with P.S. and W.v.G.; H.G., P.S., M.-A.M.-P. and L.M.T. wrote the manuscript.

Correspondence to Pattabhiraman Shankaranarayanan or Hinrich Gronemeyer.

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Competing interests

H.G., P.S. and L.M.T. have filed a patent application describing the methods presented in this study (EP11305531.3).

Supplementary information

Supplementary Fig. 1

Multidimensional ChIP-seq comparison including ChIPs done with very small numbers of cells. Four representative screenshots of ERα (purple) and RNA polymerase II (blue) profiles established from H3396 breast cancer cells2, compared with the corresponding H3K4me3 profiles (red; cumulative profiles of two biological replicates). These profiles were established by standard ChIP-seq procedure. Below the corresponding profiles derived LinDA-ChIP-seq assays done with 10,000 and 1,000 cells are displayed (black). Note the coherence between the profiles derived from 10,000 cells with the standard procedure, which is only partially maintained when 1,000 cells were used. (PDF 159 kb)

Supplementary Table 1

Quantification of yields of RNA and double-stranded DNA obtained from representative ChIPs performed with different numbers of cells and two different antibodies. LinDA was performed on ChIPs and total RNA was quantified after the in vitro transcription step; the total DNA was quantified after the final step just before Illumina sequencing. No LinDA was performed on the 2 million cell sample for ERα and the 1 million cells sample for the H3K4me3. The RNA and DNA amounts shown here for the ERα experiments correspond to the same ChIPs for which the sequencing data have been detailed in the Suppl. Table 1 of ref. 14. Note that these are independent ChIP experiments and that numbers cannot be extrapolated linearly. However, the LinDA data are representative and indicate an "apparent experimental amplification factor" between about 2,000-fold (for 5,000 cells, ERα) and 400-fold (for 100,000 cells, ERα (PDF 137 kb)

Supplementary Table 2

The antibody to cell ratios for ChIPs with small number of cells. ChIPs were performed with antibodies directed to ERa (sc-543, Santa Cruz Biotechnology) or H3K4me3 (AB8580, Abcam) using estrogen-treated H3396 breast cancer cells and the standard ChIP protocol. (PDF 124 kb)

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Shankaranarayanan, P., Mendoza-Parra, M., van Gool, W. et al. Single-tube linear DNA amplification for genome-wide studies using a few thousand cells. Nat Protoc 7, 328–339 (2012). https://doi.org/10.1038/nprot.2011.447

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