A parallelized, automated platform enabling individual or sequential ChIP of histone marks and transcription factors Riccardo Dainesea,b, Vincent Gardeuxa,b, Gerard Llimosa,b, Daniel Alperna,b, Jia Yuan Jianga, Antonio Carlos Alves Meireles-Filhoa,b, and Bart Deplanckea,b,1 aSchool of Life Sciences, Institute of Bioengineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland; and bSwiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland Edited by Bing Ren, Ludwig Institute for Cancer Research, La Jolla, CA, and accepted by Editorial Board Member Christopher K. Glass April 16, 2020 (received for review August 6, 2019) Despite its popularity, chromatin immunoprecipitation followed preamplification RNA-seq workflow consists of only three steps— by sequencing (ChIP-seq) remains a tedious (>2 d), manually in- that is, cell lysis, RNA capturing and reverse transcription—ChIP- tensive, low-sensitivity and low-throughput approach. Here, we seq typically involves several preamplification steps (cross-linking, combine principles of microengineering, surface chemistry, and lysis, fragmentation, IP, end repair, and adapter ligation). Finally, molecular biology to address the major limitations of standard any given RNA transcript is present in each cell in numerous copies, ChIP-seq. The resulting technology, FloChIP, automates and mini- which increases the likelihood of its capture and detection, whereas aturizes ChIP in a beadless fashion while facilitating the down- each locus-specific protein−DNA contact occurs a maximum of two stream library preparation process through on-chip chromatin times in a diploid cell. The combination of these idiosyncratic dif- tagmentation. FloChIP is fast (<2 h), has a wide dynamic range 6 ferences, together with the lack of enabling solutions, has thus far (from 10 to 500 cells), is scalable and parallelized, and supports prevented the ChIP-seq technology, as opposed to other NGS- antibody- or sample-multiplexed ChIP on both histone marks and ’ based methods, from reaching its full potential in terms of adop- transcription factors. In addition, FloChIP s interconnected design tion, utility, and biomedical relevance. allows for straightforward chromatin reimmunoprecipitation, In addition to the standard ChIP protocol, a modification of which allows this technology to also act as a microfluidic sequen- its workflow involving sequential ChIP has been developed to tial ChIP-seq system. Finally, we ran FloChIP for the transcription infer the genomic cooccurrence of two distinct (protein) targets. factor MEF2A in 32 distinct human lymphoblastoid cell lines, pro- In principle, sequential ChIP consists of performing ChIP twice viding insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B cell-specific gene regula- on the same input chromatin, which leads to a multiplication of tion. Together, our results validate FloChIP as a flexible and the inefficiencies mentioned above. Therefore, not only does reproducible automated solution for individual or sequential sequential ChIP show the same limitations as regular ChIP-seq, ChIP-seq. but these come in an augmented form due to its consecutive nature. As a result, few studies have so far performed sequential microfluidics | epigenetics | ChIP-seq | transcription factor Significance he genome-wide distribution and dynamics of protein−DNA Tinteractions constitute a fundamental aspect of gene regu- Chromatin immunoprecipitation followed by next-generation lation. Chromatin immunoprecipitation followed by next gener- sequencing (ChIP-seq) has become the most widely used ation sequencing (ChIP-seq) (1) has become the most widespread method to infer the genomic binding locations of chromatin- technique for mapping protein−DNA interactions genome-wide. associated proteins such as histones and transcription factors. ChIP-seq has been successfully applied to dozens of transcription Since its emergence in 2007, ChIP-seq has been performed factors (TFs), histone modifications, chromatin modifying com- through a long sequence of manual steps, with consequent plexes, and other chromatin-associated proteins in humans and limitations in throughput and sensitivity of the technique. other model organisms (2). The ENCODE (Encyclopedia of DNA Here, we present a microfluidic implementation of the ChIP Elements) and modENCODE consortia alone have already per- procedure, named FloChIP, which greatly streamlines the ex- formed more than 8,000 ChIP-seq experiments, which have greatly perimental workflow. We show that FloChIP can perform enhanced our collective understanding of how gene regulatory standard ChIP and sequential ChIP assays in parallel and re- processes are orchestrated in humans as well as several model or- producibly in an automated fashion for histone marks and ganisms (3). In addition, ChIP-seq proved to be essential to acquire transcription factors. new insights into genomic organization (4–6) and into the mecha- nisms underlying genomic variation-driven phenotypic diversity and Author contributions: R.D. and B.D. designed research; R.D. performed research; R.D., G.L., D.A., J.Y.J., and A.C.A.M.-F. contributed new reagents/analytic tools; R.D. and V.G. disease susceptibility (5, 7, 8). More specifically, this assay proved analyzed data; and R.D. and B.D. wrote the paper. crucial in determining the DNA binding properties of hundreds of The authors declare no competing interest. TFs (9). Nevertheless, in comparison to other widespread methods This article is a PNAS Direct Submission. B.R. is a guest editor invited by the based on next generation sequencing (NGS)—e.g., RNA-seq (10) Editorial Board. and assay for transposase-accessible chromatin using sequencing Published under the PNAS license. — (ATAC-seq) (11) ChIP-seq lags behind in some key metrics, that Data deposition: All data have been deposited on ArrayExpress: E-MTAB-7179 (MEF2A is, throughput, sensitivity, and automation, which hinders its wider data), E-MTAB-7150 (decreasing cell number data), E-MTAB-7186 (sequential ChIP data), adoption and reproducibility. For example, while RNA-seq can now E-MTAB-8533 (TF benchmarking data), and E-MTAB-8551 (histone mark data). be regularly performed on hundreds or thousands of single cells 1To whom correspondence may be addressed. Email: [email protected]. using readily available workflows (12, 13), ChIP-seq has largely This article contains supporting information online at https://www.pnas.org/lookup/suppl/ remained labor intensive and limited to a few samples per run, doi:10.1073/pnas.1913261117/-/DCSupplemental. each composed of millions of cells. Moreover, while a typical First published May 27, 2020. 13828–13838 | PNAS | June 16, 2020 | vol. 117 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1913261117 Downloaded by guest on September 29, 2021 ChIP followed by NGS (14, 15) (sequential ChIP-seq), and most BSA, which passively adsorbs to the hydrophobic walls of the of the available studies have so far relied on qPCR to validate microfluidic device. This layer has both an insulating role, pre- putative bivalent regions (16–18) (sequential ChIP-qPCR). venting nonspecific adsorption of chromatin to the chip walls, and a In recent years, several attempts have been made to alleviate docking role for the next layer, which is obtained by flowing on-chip some of the limitations of the ChIP-seq and sequential ChIP a solution containing neutravidin that strongly binds to the biotin approaches. Gasper et al. (19) and Aldridge et al. (20) addressed groups of the first layer. The third layer is formed by flowing a the issue of automation by implementing the manual steps of a solution of biotinylated protein A/G, which becomes firmly immo- conventional ChIP-seq workflow on robotic liquid handling sys- bilized by the unsaturated binding sites of the neutravidin layer. tems. However, in these examples, automation was balanced Protein A/G is a recombinant protein used in a variety of immu- with sensitivity, since these workflows still require tens of mil- noassays due to its ability to strongly bind to a large number of lions of cells per experiment. Van Galen et al. (21) and Chabbert different antibodies. This ability is retained by FloChIP’ssurface et al. (22) addressed the issue of throughput by barcoding and functionalization, which thus constitutes a general substrate for pooling chromatin samples before IP. Although van Galen et al. antibody pull-down. proved that their approach led to higher sensitivity (500 cells per Another critical feature of FloChIP’s workflow is the microfluidic ChIP), neither approach is automated, and both are, so far, tagmentation of immunoprecipitated chromatin. In a previous limited to the detection of histone marks. Ma et al. (23) and study, tagmentation of bead-bound chromatin (ChIPmentation) Rotem et al. (24) addressed the limitation of sensitivity with two was shown to generally increase cost-effectiveness and sensitivity of different microfluidic-based strategies. Ma et al. focused on the ChIP-seq workflow (25). Here, we built upon this concept and improving the efficiency of the IP step by confining it within adaptedittoobtainanexampleofChIPmentationperformeddi- microfluidic channels. Although they showed good IP efficiency rectly on chromatin bound to the inner walls of a microfluidic de- down to as few as 30 cells, their approach requires impractical vice (Fig. 1A, indexing and elution). Briefly, this is achieved by antibody−oligo conjugates, is not automated, and was