Preparation of Low‐Input and Ligation‐Free ChIP‐seq Libraries Using Template‐Switching Technology

Nathalie Bolduc1, Alisa P. Lehman1, Andrew Farmer1

1 Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.28
DOI:  10.1002/cpmb.24
Online Posting Date:  October, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Chromatin immunoprecipitation (ChIP) followed by high‐throughput sequencing (ChIP‐seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low‐abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP‐seq workflow that can be applied to small cell numbers, including a robust single‐tube and ligation‐free method for preparation of sequencing libraries from sub‐nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA‐binding proteins and cross‐linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross‐linking reversal and DNA recovery. Finally, we describe a fast, ligation‐free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc.

Keywords: ChIP; ChIP‐seq; template switching; SMART; NGS

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: ChIP ASSAY USING AN anti‐H3K4me3 ANTIBODY
  • Basic Protocol 2: Recover and Purify DNA (ssDNA)
  • Alternate Protocol 1: Recover and Purify DNA (dsDNA)
  • Basic Protocol 3: Prepare ChIP‐seq Libraries
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: ChIP ASSAY USING AN anti‐H3K4me3 ANTIBODY

  • Cells growing in appropriate growth medium ( appendix 3F; Phelan, )
  • Cross‐linking solution (see recipe), prepared fresh just before use
  • 2 M glycine (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS, with Ca2+ and Mg2+; Sigma, cat. no. D8662)
  • Liquid nitrogen
  • Sonication buffer (see recipe)
  • ProteoGuard EDTA‐Free Protease Inhibitor Cocktail (Clontech, cat. no. 635673)
  • ChIP dilution buffer (see recipe)
  • 1 µg/µl ChIP‐seq‐grade anti‐H3K4me3 polyclonal antibody (Diagenode, cat. no. C15410003)
  • Protein A/G PLUS‐Agarose immunoprecipitation reagent (Santa Cruz Biotechnologies, cat. no. sc‐2003)
  • ChIP wash buffers #1, #2, and #3 (see reciperecipes)
  • TE buffer, pH 8 ( appendix 22)
  • Scepter 2.0 Cell Counter (EMD Millipore, cat. no. PHCC20040 or PHCC20060)
  • Cell scrapers
  • 50‐ml conical polypropylene tubes (e.g., Corning Falcon)
  • Refrigerated tabletop centrifuge
  • Bioruptor Pico sonication device with adaptor for 0.65‐ml tubes (Diagenode, Model No. B0106001)
  • 0.65‐ml Bioruptor microtubes (Diagenode, cat. no. WA‐005‐0500)
  • Low‐retention 1.5‐ml tubes (DNA LoBind Tubes, Eppendorf, cat. no. 022431021)
  • Rotating shaker
  • Additional reagents and equipment for basic cell culture techniques ( appendix 3F; Phelan, )
CAUTION: Always perform the cross‐linking procedure inside a fume hood to avoid exposure to formaldehyde vapor.

Basic Protocol 2: Recover and Purify DNA (ssDNA)

  • ChIP Elute Kit (Clontech, cat. no. 634887) containing:
    • ChIP Elute Resin
    • Proteinase K
    • DNA Dilution Buffer
    • ssDNA Binding Buffer
    • ssDNA Binding Columns
    • Collection Tubes
    • ssDNA Wash Buffer (concentrate)
  • Washed Protein A/G beads bound to cross‐linked DNA and antibody ( protocol 1)
  • 10 mg/ml DNase‐free RNase A (optional)
  • 100% ethanol
  • Qubit ssDNA Assay Kit (Life Technologies, cat. no. Q10212)
  • Heat block capable of holding 1.5‐ml tubes
  • Low‐retention 1.5‐ml tubes (DNA LoBind Tubes, Eppendorf, cat. no. 022431021)
  • Qubit 2.0 Fluorometer (Life Technologies)

Alternate Protocol 1: Recover and Purify DNA (dsDNA)

  Additional Materials (also see protocol 2)
  • SDS elution buffer (see recipe)
  • 5 M NaCl
  • Proteinase K solution (see recipe)
  • PCR purification kit, e.g., Macherey‐Nagel NucleoSpin Gel and PCR Clean‐Up kit (Clontech, cat. no. 740609.50)
  • Buffer NTB (Clontech, cat. no. 740595.150)
  • Qubit dsDNA HS Assay Kit (Life Technologies, cat. no. Q32851)

Basic Protocol 3: Prepare ChIP‐seq Libraries

  • dsDNA or ssDNA from ChIP ( protocol 2 or protocol 3Alternate Protocol)
  • DNA SMART ChIP‐Seq Kit (Clontech, cat. nos. 634865, 634866, 634867), containing:
    • DNA Dilution Buffer (5 mM Tris·Cl, pH 8.5; also see appendix 22)
    • DNA SMART Buffer
    • Shrimp Alkaline Phosphatase
    • DNA SMART T‐Tailing Mix
    • Terminal Deoxynucleotidyl Transferase
    • DNA SMART Poly(dA) Primer
    • DNA SMART Oligonucleotide Mix
    • SMARTScribe Reverse Transcriptase
    • Indexing Primer Set HT for Illumina (12, 48 A, or 48 B)
    • SeqAmp PCR Buffer (2×)
    • SeqAmp DNA Polymerase
    • Library Elution Buffer
  • Agencourt AMPure XP PCR Purification Kit (Beckman Coulter, cat. no. A63880 or A63881)
  • 80% ethanol, made fresh for each experiment
  • Qubit dsDNA HS Assay Kit (Life Technologies, cat. no. Q32851)
  • Agilent High Sensitivity DNA Kit (Agilent, cat. no. 5067‐4626)
  • 0.2‐ml nuclease‐free thin‐wall 8‐strip PCR tubes (GeneMate, cat. no. T‐3035‐1; or USA Scientific, cat. no. 1402‐4700)
  • Thermal cycler with heated lid
  • Magnetic separation device for 0.2‐ml tubes
  • Low‐retention, nuclease‐free 1.5‐ml tubes (DNA LoBind Tubes, Eppendorf, cat. no. 022431021)
  • Qubit 2.0 Fluorometer (Life Technologies)
  • Agilent 2100 Bioanalyzer
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Adiconis, X., Borges‐Rivera, D., Satija, R., DeLuca, D.S., Busby, M.A., Berlin, A.M., Sivachenko, A., Thompson, D.A., Wysoker, A., Fennell, T., Gnirke, A., Pochet, N., Regev, A., and Levin, J.Z. 2013. Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples. Nat. Methods 10:623‐629. doi: 10.1038/nmeth.2483
  Adli, M., Zhu, J., and Bernstein, B.E. 2010. Genome‐wide chromatin maps derived from limited numbers of hematopoietic progenitors. Nat. Methods 7:615‐618. doi: 10.1038/nmeth.1478
  Bailey, T., Krajewski, P., Ladunga, I., Lefebvre, C., Li, Q., Liu, T., Madrigal, P., Taslim, C., and Zhang, J. 2013. Practical guidelines for the comprehensive analysis of ChIP‐seq data. PLoS Comput. Biol. 9:e1003326. doi: 10.1371/journal.pcbi.1003326
  Bowman, S.K., Simon, M.D., Deaton, A.M., Tolstorukov, M., Borowsky, M.L., and Kingston, R.E. 2013. Multiplexed Illumina sequencing libraries from picogram quantities of DNA. BMC Genomics 14:466. doi: 10.1186/1471‐2164‐14‐466
  Chabbert, C.D., Adjalley, S.H., Klaus, B., Fritsch, E.S., Gupta, I., Pelechano, V., and Steinmetz, L.M. 2015. A high‐throughput ChIP‐Seq for large‐scale chromatin studies. Mol. Syst. Biol. 11:777. doi: 10.15252/msb.20145776
  Chenchik, A., Zhu, Y., Diatchenko, L., Li, R., Hill, J., and Siebert, P. 1998. Generation and use of high‐quality cDNA from small amounts of total RNA by SMART PCR. In RT‐PCR Methods for Gene Cloning and Analysis (P. Siebert and J. Larrick, eds.) pp. 305‐319. BioTechniques Books, Natick, Mass.
  Feng, J., Liu, T., Qin, B., Zhang, Y., and Liu, X.S. 2012. Identifying ChIP‐seq enrichment using MACS. Nat. Protoc. 7:1728‐1740. doi: 10.1038/nprot.2012.101
  Furey, T.S. 2012. ChIP‐seq and beyond: New and improved methodologies to detect and characterize protein‐DNA interactions. Nat. Rev. Genet. 13:840‐852. doi: 10.1038/nrg3306
  Guo, Y., Mahony, S., and Gifford, D.K. 2012. High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints. PLoS Comput. Biol. 8:e1002638. doi: 10.1371/journal.pcbi.1002638
  He, Q., Johnston, J., and Zeitlinger, J. 2015. ChIP‐nexus enables improved detection of in vivo transcription factor binding footprints. Nat. Biotechnol. 33:395‐401. doi: 10.1038/nbt.3121
  Hoeijmakers, W.A., Bártfai, R., Françoijs, K.J., and Stunnenberg, H.G. 2011. Linear amplification for deep sequencing. Nat. Protoc. 6:1026‐1036. doi: 10.1038/nprot.2011.345
  Kharchenko, P.V., Tolstorukov, M.Y., and Park, P.J. 2008. Design and analysis of ChIP‐seq experiments for DNA‐binding proteins. Nat. Biotechnol. 2612:1351‐1359. doi: 10.1038/nbt.1508
  Kharchenko, P.V., Alekseyenko, A.A., Schwartz, Y.B., Minoda, A., Riddle, N.C., Ernst, J., Sabo, P.J., Larschan, E., Gorchakov, A.A., Gu, T., Linder‐Basso, D., Plachetka, A., Shanower, G., Tolstorukov, M.Y., Luquette, L.J., Xi, R., Jung, Y.L., Park, R.W., Bishop, E.P., Canfield, T.K., Sandstrom, R., Thurman, R.E., MacAlpine, D.M., Stamatoyannopoulos, J.A., Kellis, M., Elgin, S.C., Kuroda, M.I., Pirrotta, V., Karpen, G.H., and Park, P.J. 2011. Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471:480‐485. doi: 10.1038/nature09725
  Landt, S.G., Marinov, G.K., Kundaje, A., Kheradpour, P., Pauli, F., Batzoglou, S., Bernstein, B.E., Bickel, P., Brown, J.B., Cayting, P., Chen, Y., DeSalvo, G., Epstein, C., Fisher‐Aylor, K.I., Euskirchen, G., Gerstein, M., Gertz, J., Hartemink, A.J., Hoffman, M.M., Iyer, V.R., Jung, Y.L., Karmakar, S., Kellis, M., Kharchenko, P.V., Li, Q., Liu, T., Liu, X.S., Ma, L., Milosavljevic, A., Myers, R.M., Park, P.J., Pazin, M.J., Perry, M.D., Raha, D., Reddy, T.E., Rozowsky, J., Shoresh, N., Sidow, A., Slattery, M., Stamatoyannopoulos, J.A., Tolstorukov, M.Y., White, K.P., Xi, S., Farnham, P.J., Lieb, J.D., Wold, B.J., and Snyder, M. 2012. ChIP‐seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 22:1813‐1831. doi: 10.1101/gr.136184.111
  Lara‐Astiaso, D., Weiner, A., Lorenzo‐Vivas, E., Zaretsky, I., Jaitin, D.A., David, E., Keren‐Shaul, H., Mildner, A., Winter, D., Jung, S., Friedman, N., and Amit, I. 2014. Chromatin state dynamics during blood formation. Science 345:943‐949. doi: 10.1126/science.1256271
  Mundade, R., Ozer, H.G., Wei, H., Prabhu, L., and Lu, T. 2014. Role of ChIP‐seq in the discovery of transcription factor binding sites, differential gene regulation mechanism, epigenetic marks and beyond. Cell Cycle 13:2847‐2852. doi: 10.4161/15384101.2014.949201
  Ongena, K., Das, C., Smith, J.L., Gil, S., and Johnston, G. 2010. Determining cell number during cell culture using the scepter cell counter. J. Vis. Exp. 45:2204. doi:10.3791/2204
  Peng, X., Wu, J., Brunmeir, R., Kim, S.Y., Zhang, Q., Ding, C., Han, W., Xie, W., and Xu, F. 2014. TELP, a sensitive and versatile library construction method for next‐generation sequencing. Nucleic Acids Res. 43:e35. doi: 10.1093/nar/gku818
  Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18. doi: 10.1002/0471142727.mba03fs74
  Raha, D., Hong, M., and Snyder, M. 2010. ChIP‐Seq: A method for global identification of regulatory elements in the genome. Curr. Protoc. Mol. Biol. 91:21.19.1‐21.19.14. doi: 10.1002/0471142727.mb2119s91
  Rhee, H.S. and Pugh, B.F. 2012. ChIP‐exo method for identifying genomic location of DNA‐binding proteins with near‐single‐nucleotide accuracy. Curr. Protoc. Mol. Biol. 100:21.24.1‐21.24.14. doi: 10.1002/0471142727.mb2124s100
  Robertson, G., Hirst, M., Bainbridge, M., Bilenky, M., Zhao, Y., Zeng, T., Euskirchen, G., Bernier, B., Varhol, R., Delaney, A., Thiessen, N., Griffith, O.L., He, A., Marra, M., Snyder, M., and Jones, S. 2007. Genome‐wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat. Methods 4:651‐657. doi: 10.1038/nmeth1068
  Rozowsky, J., Euskirchen, G., Auerbach, R.K., Zhang, Z.D., Gibson, T., Bjornson, R., Carriero, N., Snyder, M., and Gerstein, M.B. 2007. PeakSeq: Systematic scoring of ChIP‐Seq experiments relative to controls. Nat. Biotechnol. 27:66‐75. doi: 10.1038/nbt.1518.
  Schmidl, C., Rendeiro, A.F., Sheffield, N.C., and Bock, C. 2015. ChIPmentation: Fast, robust, low‐input ChIP‐seq for histones and transcription factors. Nat. Methods 12:963‐965. doi: 10.1038/nmeth.3542.
  Serandour, A.A., Brown, G.D., Cohen, J.D., and Carroll, J.S. 2013. Development of an Illumina‐based ChIP‐exonuclease method provides insight into FoxA1‐DNA binding properties. Genome Biol. 14:R147. doi: 10.1186/gb‐2013‐14‐12‐r147
  Shankaranarayanan, P., Mendoza‐Parra, M.A., Walia, M., Wang, L., Li, N., Trindade, L.M., and Gronemeyer, H. 2011. Single‐tube linear DNA amplification (LinDA) for robust ChIP‐seq. Nat. Methods 8:565‐567. doi: 10.1038/nmeth.1626
  Shanker, S., Paulson, A., Edenberg, H.J., Peak, A., Perera, A., Alekseyev, Y.O., Beckloff, N., Bivens, N.J., Donnelly, R., Gillaspy, A.F., Grove, D., Gu, W., Jafari, N., Kerley‐Hamilton, J.S., Lyons, R.H., Tepper, C., and Nicolet, C.M. 2015. Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA. J. Biomol. Tech. 26:4‐18. doi: 10.7171/jbt.15‐2601‐001
  Teytelman, L., Thurtle, D.M., Rine, J., and van Oudenaarden, A. 2013. Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc. Natl. Acad. Sci. U.S.A. 110:18602‐18607. doi: 10.1073/pnas.1316064110
  Turchinovich, A., Surowy, H., Serva, A., Zapatka, M., Lichter, P., and Burwinkel, B. 2014. Capture and amplification by tailing and switching (CATS). An ultrasensitive ligation‐independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA. RNA Biol. 11:817‐828. doi: 10.4161/rna.29304
  Wu, A.R., Neff, N.F., Kalisky, T., Dalerba, P., Treutlein, B., Rothenberg, M.E., Mburu, F.M., Mantalas, G.L., Sim, S., Clarke, M.F., and Quake, S.R. 2014. Quantitative assessment of single‐cell RNA‐sequencing methods. Nat. Methods 11:41‐46. doi: 10.1038/nmeth.2694
PDF or HTML at Wiley Online Library