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
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Abstract

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

     
 
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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
     
 
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Materials

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

  Materials
  • 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)

  Materials
  • 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

  Materials
  • 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
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Figures

Videos

Literature Cited

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