Identifying Chromosomal Targets of DNA‐Binding Proteins by Sequence Tag Analysis of Genomic Enrichment (STAGE)

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Sequence Tag Analysis of Genomic Enrichment (STAGE) is a method for experimentally identifying the in vivo chromosomal targets of DNA?binding proteins in any sequenced genome. STAGE generates 21?bp tags derived from DNA isolated by chromatin immunoprecipitation (ChIP; UNIT 21.3 ). Concatamers of tags are cloned and sequenced to yield a STAGE library. Tags in the library represent DNA fragments that were occupied by the DNA?binding protein, and mapping these tag sequences to the genome identifies the binding loci of the DNA?binding protein in vivo. STAGE can be applied to any sequenced genome to identify targets of DNA?binding proteins without requiring extensive microarray resources.

Keywords: Chromatin immunoprecipitation (ChIP); DNA?protein interaction; Serial analysis of gene expression (SAGE); Sequence tag analysis of genomic enrichment (STAGE); Tags

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Basic Protocol 1: Sequence Tag Analysis of Genomic Enrichment (STAGE)
Support Protocol 1: Subtraction STAGE (SubSTAGE)
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Sequence Tag Analysis of Genomic Enrichment (STAGE)

Materials

10 ng/µl chromatin immunoprecipitated (ChIP) DNA fragments (unit 21.3 ; test enrichment by PCR as described in that unit; if not sufficiently high, perform subtraction as described in protocol 2 to improve enrichment)
5× Sequenase reaction buffer (USB)
40 µM round A primer (see recipe for PCR primers)
10 ng/µl sheared genomic DNA generated from the same cells used in the ChIP procedure
TE buffer, pH 8.0 ( appendix 22 )
Round A dNTP mix: 3 mM each dNTP (A, C, G, and T) in TE buffer, pH 8.0
0.1 M dithiothreitol (DTT)
0.5 mg/ml bovine serum albumin (BSA)
13 U/µl Sequenase (USB)
10× PCR buffer (see recipe )
25 mM MgCl₂
500 µM biotinylated round B primer (see recipe for PCR primers)
Standard dNTP mix: 25 mM each dNTP (A, C, G, and T) in TE buffer, pH 8.0
5 U/µl Taq DNA polymerase
1% and 1.5% agarose gels (unit 2.5 )
PC8 (see recipe )
20 mg/ml glycogen (Roche Diagnostics)
7.5 M ammonium acetate (Sigma)
100% and 70% ethanol
LoTE buffer (see recipe )
100× BSA (New England Biolabs)
10× Buffer 4 (New England Biolabs)
Nla III restriction endonuclease (New England Biolabs; also see unit 3.1 )
Dynabeads M‐280 Streptavidin slurry (Dynal)
1× and 2× BW buffer (see recipe )
Annealed linkers 1 and 2: linkers 1A, 1B, 2A, and 2B (see recipe ), phosphorylated and annealed as in Support Protocol 3 of unit 25.6
10× ligase buffer (New England Biolabs)
T4 DNA ligase (high concentration, 2000 U/µl; New England Biolabs)
32 mM S ‐adenosylmethionine (SAM; New England Biolabs)
Mme I restriction endonuclease (New England Biolabs)
Primers 1 and 2 (see recipe for PCR primers)
12% (10‐well) and 8% nondenaturing polyacrylamide gels prepared in TBE buffer (unit 2.7 )
10‐bp and 1‐kbp DNA ladders
6× sample loading buffer (see recipe )
1 µg/µl pZErO‐1 plasmid (Invitrogen), linearized
10× Buffer 2 (New England Biolabs)
Sph I restriction endonuclease (New England Biolabs)
ElectroMAX DH10B electroporation‐competent cells (Invitrogen; frozen at −80°C)
SOC medium (see recipe )
10‐cm Zeocin‐containing low‐salt LB plates (see recipe )
Dimethyl sulfoxide (DMSO)
5 µM M13 forward and reverse primers (see recipe for PCR primers)

0.2‐ml thin‐walled PCR tubes
Thermal cycler
0.5‐ and 1.5‐ml snap‐cap and 2‐ml screw‐cap microcentrifuge tubes
Magnetic rack for Dynabead separations (Dynal)
12°, 16°, 37°, 42°, 50°, and 65°C water baths or temperature blocks
15‐ and 50‐ml conical tubes
Tabletop centrifuge with swinging‐bucket rotor
25‐G needles
Spin‐X centrifuge tube filters (Costar)
Bio‐Rad GenePulser electroporator (or equivalent) and 0.1‐mm disposable electroporation cuvettes (Bio‐Rad)
Platform shaker
37° and 30°C incubators
96‐well PCR plate (MJ Research)
Sterile toothpicks

Additional reagents and equipment for PCR (unit 15.1 ), restriction enzyme digestion of DNA (unit 3.1 ), phenol extraction and ethanol precipitation of DNA (unit 2.1 ), agarose gel electrophoresis (unit 2.5 ), nondenaturing polyacrylamide gel electrophoresis (unit 2.7 ), ligation (unit 3.14 ), electroporation of bacteria (unit 1.8 ), and direct DNA sequencing of PCR products (unit 15.2 )

NOTE: Use aerosol‐barrier pipet tips to prevent contamination of PCR reactions; see appendix 22 for additional precautions with PCR.

Support Protocol 1: Subtraction STAGE (SubSTAGE)

Unbiotinylated round B primer (see recipe for PCR primers; omit biotinylation)
1 M Tris⋅Cl, pH 7.5 ( appendix 22 )
0.5 M EDTA ( appendix 22 )
1 M NaOH ( appendix 22 )
72° and 95°C water baths or heating blocks

Additional reagents and equipment for ChIP‐PCR (unit 21.3 )

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Figures

Figure 21.10.1 Scheme for sequence tag analysis of genomic enrichment (STAGE).

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Literature Cited

Literature Cited
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