Capture Hybridization Analysis of RNA Targets (CHART)

Matthew D. Simon1

1 Department of Molecular Biophysics and Biochemistry and Chemical Biology Institute, Yale University, West Haven, Connecticut
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 21.25
DOI:  10.1002/0471142727.mb2125s101
Online Posting Date:  January, 2013
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Abstract

The genome is regulated by trans‐acting factors that bind to specific loci in chromatin. In addition to protein factors, it has become clear that large non‐coding RNAs can also act on chromatin at sites distant from where they are transcribed. This unit describes a means of identifying the genomic targets of those large non‐coding RNAs. To accomplish this, the endogenous RNA of interest (here Drosophila roX2 is used as an example) is enriched from cross‐linked chromatin extracts using short biotinylated complementary oligodeoxyribonucleotides. The targets of the RNA can be determined by examining the proteins and DNA that are enriched under these conditions. This analysis can be extended genome‐wide by subjecting the enriched DNA to deep sequencing. Curr. Protoc. Mol. Biol. 101:21.25.1–21.25.16. © 2013 by John Wiley & Sons, Inc.

Keywords: CHART; chromatin; non‐coding RNA; lncRNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing Cross‐Linked Nuclei
  • Basic Protocol 2: Design of Capture Oligonuleotides that Target Accessible Regions of the RNA
  • Basic Protocol 3: Performing CHART Enrichment
  • Basic Protocol 4: Preparation of Target DNA, RNA, and Proteins from CHART Enrichment
  • Support Protocol 1: Analysis of CHART‐Enriched Proteins
  • Support Protocol 2: Quality Control of CHART DNA Enrichment by qPCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparing Cross‐Linked Nuclei

  Materials
  • Drosophila S2 cells (ATCC, cat. no. CRL‐1963)
  • CCM3 medium (Hyclone, cat. no. SH30065.02)
  • Penicillin‐streptomycin mixture (e.g., Lonza, cat. no. 12001‐350)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 16% (w/v) formaldehyde (10 ml ampules, Thermo Scientific, cat. no. 28908)
  • Liquid N 2
  • Sucrose buffer (see recipe), ice cold
  • Glycerol buffer (see recipe)
  • 500‐ml shaker flasks
  • 27°C incubator without added CO 2: e.g., New Brunswick Scientific Excella E24 Incubator Shaker
  • End‐over‐end rotator
  • Refrigerated centrifuge
  • Glass Dounce homogenizer with tight pestle (15 ml)
  • 15‐ml conical tubes (e.g., BD Falcon)

Basic Protocol 2: Design of Capture Oligonuleotides that Target Accessible Regions of the RNA

  Materials
  • Nuclei pellet (109 S2 nuclei or 108 mammalian nuclei from protocol 1)
  • Nuclei wash buffer (see recipe)
  • Sonication buffer (see recipe)
  • Liquid N 2
  • 1 M MgCl 2
  • 1 M dithiothreitol (DTT)
  • 5 U/µl RNase H (NEB, cat. no. M0297L)
  • 20 U/µl SUPERasIN (Ambion, cat. no. AM2696)
  • 20‐mer oligonucleotides (IDT; for more information see Step 11.
  • RQ1 DNase (Promega, cat. no. M6101)
  • 1 M CaCl 2
  • Quenching buffer (see recipe)
  • PureLink Micro‐to‐Midi Total RNA Purification System (Invitrogen, cat. no, 12183‐018)
  • SuperScript VILO cDNA Synthesis Kit (Invitrogen, cat. no. 11754‐050)
  • iTaq UniverSYBR Green (Bio‐Rad, cat. no. 172‐5120)
  • Appropriate qPCR primer sets
  • Refrigerated centrifuge
  • Covaris S2 instrument (or other similar means of shearing DNA)
  • 1.7‐ml microcentrifuge tubes
  • PCR tubes in strips of 8
  • Thermal cycler
  • ABI 7500 RT‐PCR instrument or equivalent
  • NanoDrop spectrophotometer (Thermo Scientific; also see appendix 3J)
  • Additional reagents and equipment for microvolume quantitation of nucleic acids using a NanoDrop spectrophotometer ( appendix 3J)

Basic Protocol 3: Performing CHART Enrichment

  Materials
  • Nuclei pellet from 1 × 109 cells ( protocol 1)
  • Phosphate‐buffered saline (PBS, appendix 22), pH 7.4
  • 16% (w/v) formaldehyde (10‐ml ampules, Thermo Scientific, cat. no. 28908)
  • Wash Buffer 100 (WB100; see recipe)
  • 20 U/µl SUPERasIN (Ambion, cat. no. AM2696)
  • Roche complete EDTA‐free protease inhibitor cocktail (resuspend each tablet in 500 µl H 2O to provide a 100× working solution)
  • Liquid N 2
  • 1 M dithiothreitol (DTT)
  • Denaturant buffer (see recipe)
  • 2× hybridization buffer (see recipe)
  • 20 µM CHART capture oligo cocktail ( protocol 2)
  • MyOne Dynabeads C1 (Invitrogen, cat. no. 650.02)
  • Wash buffer 250 (WB250): see recipe for WB100, but use 250 mM NaCl
  • RNase H elution buffer (HEB; see recipe)
  • 5 U/µl RNase H (NEB, cat. no. M0297L)
  • End‐over‐end or spinning rotator
  • 15‐ and 50‐ml conical centrifuge tubes (BD Falcon)
  • Sonicator (micro‐tip type, e.g., Misonix 3000, http://www.misonix.com/)
  • Dynal magnets for 1.7‐ml tubes
  • Dynal magnetic stand

Basic Protocol 4: Preparation of Target DNA, RNA, and Proteins from CHART Enrichment

  Materials
  • CHART‐enriched eluate ( protocol 3)
  • Nucleic Acid XLR Buffer (see recipe)
  • 20 mg/ml proteinase K (Ambion, AM2548)
  • Phenol:CHCl 3:isoamyl alcohol, 25:24:1, saturated with 10 mM Tris⋅Cl, pH 8.0/1 mM EDTA (Sigma, cat. no. P3803)
  • CHCl 3 (Fluka, cat. no. 25668)
  • 3 M sodium acetate, pH 5.5 ( appendix 22)
  • 100% and 70% ethanol
  • 10 mM Tris⋅Cl, pH 8.0 ( appendix 22)
  • PureLink Micro‐to‐Midi Total RNA Purification System (Invitrogen, cat. no. 12183‐018)
  • VILO reverse‐transcription cDNA synthesis kit (Invitrogen, cat. no. 11754‐050)
  • iTaq UniverSYBR Green (Bio‐Rad, cat. no. 172‐5120)
  • Appropriate primer sets: these primers should include at least one set that are specific to the target RNA, but do not contain the sequence targeted by the capture oligo; the region of the RNA targeted by the capture oligo will be digested by RNase H during CHART enrichment—also include at least one primer set for an abundant but unrelated RNA
  • 55° and 65°C water baths or heat blocks
  • Phase‐Lock tubes (5Prime, cat. no. 2302800)
  • Centrifuge
  • MicroTube (6 × 16 mm), AFA fiber with snap‐cap, round bottom, glass (Covaris, cat. no. 520045)
  • Covaris S2 instrument
  • ABI 7500 qPCR Instrument

Support Protocol 1: Analysis of CHART‐Enriched Proteins

  Materials
  • CHART‐enriched eluate ( protocol 3)
  • Protein XLR Buffer (see recipe)
  • Lane Marker Non‐Reducing Sample Buffer (Pierce, cat. no. 39001)
  • PCR tubes
  • PCR thermal cycler
  • Additional reagents and equipment for immunoblotting (western blotting; unit 10.8)

Support Protocol 2: Quality Control of CHART DNA Enrichment by qPCR

  Materials
  • iTaq UniverSYBR Green (Bio‐Rad, cat. no. 172‐5120)
  • iTaq SYBR Green Supermix with ROX (Bio‐Rad, cat. no. 172‐5850)
  • ABI 7500 qPCR Instrument
  • Appropriate primer sets
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Figures

Videos

Literature Cited

Literature Cited
   Carter, D., Chakalova, L., Osborne, C.S., Dai, Y.F., and Fraser, P. 2002. Long‐range chromatin regulatory interactions in vivo. Nat. Genet. 32:623‐626.
   Chu, C., Qu, K., Zhong, F.L., Artandi, S.E., and Chang, H.Y. 2011. Genomic maps of long noncoding RNA occupancy reveal principles of RNA‐chromatin interactions. Mol. Cell 44:667‐678.
   Koziol, M.J. and Rinn, J.L. 2010. RNA traffic control of chromatin complexes. Curr. Opin. Genet. Dev. 20:142‐148.
   Mariner, P.D., Walters, R.D., Espinoza, C.A., Drullinger, L.F., Wagner, S.D., Kugel, J.F., and Goodrich, J.A. 2008. Human Alu RNA is a modular transacting repressor of mRNA transcription during heat shock. Mol. Cell 29:499‐509.
   Simon, M.D., Wang, C.I., Kharchenko, P.V., West, J.A., Chapman, B.A., Alekseyenko, A.A., Borowsky, M.L., Kuroda, M.I., and Kingston, R.E. 2011. The genomic binding sites of a noncoding RNA. Proc. Natl. Acad. Sci. U.S.A. 108:20497‐20502.
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