ChIP-Chip E. coli

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{{back to protocols}}
'''This protocol is in developement and currently under investigation in the lab. Please contibute and watch changes in the next weeks!'''
'''This protocol is in developement and currently under investigation in the lab. Please contibute and watch changes in the next weeks!'''
==Curators==
==Curators==
-
'''[[User:Torsten Waldminghaus|Torsten Waldminghaus]] 06:29, 17 March 2008 (CDT)'''
+
'''[[User:Torsten Waldminghaus|Torsten Waldminghaus]]'''
''Anyone should feel free to add themselves as a curator for this consensus protocol.  You do not need to be a curator in order to contribute.  The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]].  Please contribute.''
''Anyone should feel free to add themselves as a curator for this consensus protocol.  You do not need to be a curator in order to contribute.  The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators|discussing the idea of protocol curators]].  Please contribute.''
==Abstract==
==Abstract==
-
ChIP-Chip stands for chromatine immuno precipitation and Chip in the sence of DNA-array. It is a technique to determin the DNA binding sites of a DNA binding protein on a global scale. While the basic priciple is the same there are some differences in handling cells from various organisms. This protocol is developed and tested for '''E. coli'''. It should work the same way for other bacteria but that remains to be proven (please add any experience). The main difference to other protocols is that no amplification is done after reversion of cross-linking. The basic procedure is described in <cite>Grainger2006NAR</cite>.
+
ChIP-Chip stands for <u>Ch</u>romatin <u>I</u>mmuno<u>p</u>recipitation and chip in the sense of DNA microarray. It is a technique to determine the genome-wide binding sites of a DNA-binding protein. While the basic principle is the same for all species, there are some differences in handling cells. This protocol is developed and tested for '''''E. coli'''''. It should work the same way for other bacteria but that remains to be proven. Published protocols also exist for other bacterial species, including <i>Bacillus subtilis</i>, <i>Caulobacter crescentus</i> and <i>Mycobacterium tuberculosis</i>.
-
Other ChIP-Chip protocols can be foun here:
+
Because <i>E. coli</i> can be grown to high cell densities relative to eukaryotes, it is possible to generate sufficient DNA to label without using a PCR-based method. This method uses strand displacement primer extension with Klenow DNA polymerase and amplifies the DNA ~10-fold, while simultaneously incorporating dye-coupled nucleotide.
 +
 
 +
Related protocols can be found here:
*[[Silver: ChIP-chip]]
*[[Silver: ChIP-chip]]
*[[Drosophila CHiP on chip]]
*[[Drosophila CHiP on chip]]
*[[ChIP/Chip]]
*[[ChIP/Chip]]
 +
*[[Etchevers:ChIP francais]]
 +
*[[Griffin: Ultimate Immunoprecipitation Guide]]
==Materials==
==Materials==
-
*Ultralink protein A/G beads (catalog number 53132, [http://www.piercenet.com/ Pierce])
+
*2 ml Ultralink protein A/G beads (catalog number 53132, [http://www.piercenet.com/ Pierce])
*Specific antibody for example RNA polymerase β subunit from [http://www.neoclone.com/productcatalog/index.php Neoclone, Madison]
*Specific antibody for example RNA polymerase β subunit from [http://www.neoclone.com/productcatalog/index.php Neoclone, Madison]
===Reagents===
===Reagents===
-
Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
 
-
===Equipment===
 
-
Any equipment used to perform the protocol (link to a method for using them).
 
 +
Material for about 50 chromatin immunoprecipitations:
 +
 +
*75 ml Formaldehyde (37%)
 +
*500 ml 2.5 M [[Glycine]]
 +
*1.5 l [[TBS]] (400 ml 5xTBS)
 +
*50 ml [[TE]]
 +
*20 mg/ml [[Proteinase K]] in TBS (store up to 1 year at -20 C)
 +
*1 ml 100mM [[PMSF]] or 0.5 ml 250 mM [[Pefabloc]]
 +
 +
 +
{| border="3"
 +
!Final concentration !! For 50ml
 +
|+ '''Lysis Buffer'''
 +
|-
 +
|10mM Tris (pH 8.0)||500μL 1M
 +
|-
 +
|20% sucrose||10g
 +
|-
 +
|50mM NaCl||500μL 5M
 +
|-
 +
|10mM EDTA||1mL 0.5M
 +
|-
 +
|10 mg/ml lysozyme||0.5g
 +
|-
 +
|}
 +
*Note: Prepare the 50ml without lysozyme and than add the corresponing ammount to 10 ml aliquotes and store them ad -20°C
 +
 +
 +
{| border="3"
 +
!Final concentration !! For 500ml
 +
|+ '''IP-Buffer (Immunoprecipitation Buffer)'''
 +
|-
 +
|50 mM HEPES-KOH pH 7.5||50ml 0.5M
 +
|-
 +
|150 mM NaCl||15ml 5M
 +
|-
 +
|1 mM EDTA||1ml 0.5M
 +
|-
 +
|1% Triton X 100||5ml
 +
|-
 +
|0.1 % Sodium deoxycholate||0.5g
 +
|-
 +
|0.1 % SDS||5ml 10%
 +
|-
 +
|}
 +
 +
 +
*IP-Buffer with 500 mM NaCl: add 2.8mL 5M NaCl to 40mL IP-buffer
 +
 +
 +
{| border="3"
 +
!Final concentration !! For 50ml
 +
|+ '''Wash-Buffer'''
 +
|-
 +
|10mM Tris pH 8.0||500µl 1M
 +
|-
 +
|250 mM LiCl||5 ml 2.5M
 +
|-
 +
|1 mM EDTA||100µl 0.5M
 +
|-
 +
|0.5% Nonidet-P40 (=Triton X114)||250µl
 +
|-
 +
|0.5% Sodium deoxycholate||2.5ml 10%
 +
|-
 +
|}
 +
 +
 +
{| border="3"
 +
!Final concentration !! For 10ml
 +
|+ '''Elution-Buffer'''
 +
|-
 +
|50 mM Tris (pH 7.5)||500μL 1M
 +
|-
 +
|10 mM EDTA||200μL 0.5M
 +
|-
 +
|1% SDS||1mL 10%
 +
|-
 +
|ddH<sub>2</sub>O||8.3mL
 +
|-
 +
|}
 +
 +
 +
'''For labeling the DNA:'''
 +
*BioPrime kit Invitrogen (18094-011)[http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen;PRODUCTSJSESSIONID=WK2wLRsfYChBXCWy6SLPl3J2RLYqKQWQYGbVtSMTX4XCw5lNRykr!-1264264492!496425866?cmd=catProductDetail&entryPoint=adirect&productID=18094011&CID=Search-18094-011&messageType=catProductDetail&showAddButton=true]
 +
*Cy3-dCTP GE Healthcare (PA53021)[http://www1.gelifesciences.com/aptrix/upp00919.nsf/content/WD:PA53021%20Cy3-dCT(161083530-G350)]
 +
*Cy5-dCTP GE Healthcare (PA55021) [http://www1.gelifesciences.com/aptrix/upp00919.nsf/Content/WD%3APA55021+Cy5-dCT%28161085054-G350%29?OpenDocument&hometitle=search]
 +
*Deoxynucleotides (2mM dATP, 2mM dGTP, 2mM dTTP, 0.5mM dCTP mix). Prepared as follows:
 +
**4μl 100mM dATP GE Healthcare (27-2035-01)
 +
**4μl 100mM dGTP
 +
**4μl 100mM dTTP
 +
**1μl 100mM dCTP
 +
**187μl Water
 +
**Store at -20 °C
 +
*QIAquick PCR purification columns Qiagen (28104)[http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/QIAquickPCRPurificationKit.aspx?r=1745]
 +
 +
'''Arrays'''
 +
*Oxfod Gene Technology (ogt) provides good arrays in 4*44k format compatibel with Agilent scanner and hybridisation equipment [http://www.ogt.co.uk/Prokaryotic.htm]
 +
 +
*'''Hybridisation buffer materials:'''
 +
*5M Sodium chloride Sigma (S6316)
 +
*12x MES prepared as follows:
 +
**3.52g MES free acid monohydrate Sigma (M2933)
 +
**9.66g MES Sodium salt Sigma (M3058)
 +
**Water to 40ml
 +
**pH to 6.5 with 1M Hydrochloric acid
 +
**Add water to 50ml
 +
**Filter sterilise with a 0.45μm filter
 +
**Store at 4 °C
 +
*10% Triton prepared as follows:
 +
**1ml Triton X100 Sigma (T8787)
 +
**9ml water
 +
**Store at room temperature
 +
*100% Formamide Sigma (F9037-100ml)
 +
*0.5M EDTA Sigma (E7889)
 +
*20x SSPE Sigma (S2015)
 +
*10% N-Lauroylsarcosine prepared as follows:
 +
**1g N-Lauroylsarcosine Sodium salt Sigma (L9150-100g)
 +
**Water to 10ml
 +
**Store at room temperature
 +
*Polyethylene glycol (PEG) 200 Sigma (Fluka) (88440)
 +
 +
===Equipment===
 +
*DNA Microarray Hybridization Chamber - SureHyb from Agilent [http://www.chem.agilent.com/Scripts/PDS.asp?lPage=11373]
 +
*DNA Microarray Hybridization Oven from Agilent [http://www.chem.agilent.com/Scripts/PDS.asp?lPage=18393]
 +
*DNA Microarray Scanner from Agilent [http://www.chem.agilent.com/Scripts/PDS.asp?lPage=398]
==Procedure==
==Procedure==
-
'''1. Day'''
+
This protocol has been broken up into 3 "days" but for <i>E. coli</i> it is possible to perform the entire ChIP experiment in a single day as well as the amplification/labeling step (which can be done overnight).
 +
 
   
   
''Formaldehyde cross link and sonication:''
''Formaldehyde cross link and sonication:''
Line 43: Line 171:
*Wash twice in cold 10 ml TBS (20mM; see Material) pH7.5
*Wash twice in cold 10 ml TBS (20mM; see Material) pH7.5
 +
 +
[[Image:Pause point.png]] You can freeze the cell pellet and proceed later
*Resuspend in 1 ml Lysis-Buffer  
*Resuspend in 1 ml Lysis-Buffer  
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*Add 50 μl of 100mM [[PMSF]] => final concentration of 1mM
*Add 50 μl of 100mM [[PMSF]] => final concentration of 1mM
-
*Sonicate 12 times 30 sec on ice with 30 sec breaks between sonication steps and 2 1/2 minutes breaks after the 6th and 9th step.  
+
[[Image:Critical step.png]] Sonicate so that DNA fragments are <1 kb on average (see "critical steps" section below). Using a Branson 450 sonifier with a microtip probe (inserted in the sample) 2 cycles of 30 s sonication are sufficient. Samples should be kept on ice at all times as they warm up considerably during sonication. It is important to leave the samples for at least 1 minute on ice between each round of sonication. Note that sonication parameters will vary according to the sonicator and probe used. It is possible to use a cup-horn sonicator but this requires much longer sonication and it is important to ensure that sonication is even for all samples being sonicated.
-
'''Note''': for sonication everybody, every sonicator, every day and everything else is different so see critical steps (below) and than try what work best for you.
+
-
*Centrifuge 12.000 g, 4°C, 10 min  
+
*Centrifuge 12,000 g, 4 °C, 10 min  
-
''ChIP:''
+
''IP:''
-
*Use 800 μl aliquot as input sample for one immunoprecipitation experiment
+
*Use 800 μl aliquot for one immunoprecipitation experiment
-
*Add 20 μl of Ultralink protein A/G beads  
+
*Add 20 μl of a 50% slurry of protein A sepharose or protein A/G beads (note that the beads should be selected to work with the antibody being used)
-
*Add specific antibody (for example 5 μl of RNA polymerase β subunit; see Material above)
+
*Add specific antibody (for example 1 μl of RNA polymerase β subunit; see Material above)
-
*Incubate at 4 °C over night on a slow rotator
+
*Incubate at 4 °C overnight on a slow rotator or at room temperature for 90 minutes. Note that most antibodies work fine at room temperature but some require overnight incubation at 4 °C. This has to be determined empirically, as does the amount of antibody required for the IP.
-
'''2. Day'''
+
*Collect sephareose beads by centrifugation for 1 min at 3500 rpm
-
*Collect each sample on Spin-x column by centrifugation for 2 min at 3500 rpm
+
*Pipett off supernatant and save as '''control DNA'''
-
*All following wash steps should be on a rotator at room temperature for 3min with 2 min centrifugation as above:
+
*All following wash steps should be on a rotator at room temperature for 3 min with 2 min centrifugation as above:
#Wash twice with 500 μl I-Buffer
#Wash twice with 500 μl I-Buffer
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#Wash with 500 μl TE  
#Wash with 500 μl TE  
-
*Place filter unit containing the beads into a new 1.5-ml microcentrifuge tube and add 100 μl of elution buffer. Gently pipet up and down two or three times in order to dislodge beads from the filter. Incubate 10 min in a 65 °C water bath. A water bath is used instead of other heating apparatuses in order to improve heat transfer. Make sure the sample in the tube is actually under the water and not in the air above.
+
*Add 100 μl of elution buffer. Gently pipet up and down two or three times in order to dislodge beads from the filter. Incubate 10 min in a 65 °C water bath. A water bath is used instead of other heating apparatuses in order to improve heat transfer.
-
*Centrifuge beads 2 min at 3500 rpm, room temperature. Discard filter with
+
*Centrifuge beads 1 min at 3500 rpm, room temperature.Transfer supernatant into new 1.5 ml tube.
-
beads. Transfer the eluate into new 1.5 ml tube and leave on ice until you start the next step short before you go home for dinner.
+
-
*Add 80 μl TE and 20 μl Pronase in TBS to the tube and divede on PCR tubes fitting your PCR machine (for example 2x100μl).
+
*Add 80 μl TE and 1µl RNase A (20mg/ml) to the eluted DNA and to 100µl of the control DNA (see above)
-
*To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. Store samples at 4 °C until use.
+
*Incubate for 90 min at 42°C
 +
* add 20 μl Proteinase K to each tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl). (Note: many protocols no longer have this step; I get equivalent data when I skip this)
-
'''3. Day'''
+
*To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. If not using Proteinase K, incubate overnight at 65 °C, or boil samples for 10 minutes.
-
*Purify DNA by phenol extraction and ethanol precipitation
+
*Purify DNA by phenol extraction and ethanol precipitation or using a PCR purification kit (e.g. from Qiagen)
-
*Dilute in 20 μl A. dest
+
*Elute or resuspend in 10-20 ul water
-
*Measure DNA-content idealy at a NanoDrop (should be around 0.2 to 0.4 μg)
+
*Measure DNA-content ideally at a NanoDrop (should be around 0.2 to 0.4 μg)
-
*Use 1 and 10 ng DNA as template for quantitative PCR with primers that are specific for a known binding site of your DNA-binding protein and one negative control.
+
*Use 1 and 10 ng DNA as template for quantitative PCR with primers that are specific for a known binding site of your DNA-binding protein and one negative control
 +
'''Labeling for hybridisation to microarray:'''
 +
*The chromatin immunoprecipitated DNA samples should be in 20μl volume at a concentration of approximately 20ng/μl (more is also fine).
 +
*Note that one allways needs one test sample that will be labeld with one CyDye and a control labeld with another. As control one could use chromosomal DNA or DNA immunoprecipitated from a mutant strain of the DNA binding protein of choice or from different growth conditions etc.
 +
*add 20μL 2.5x Random primer (BioPrime kit) to each 20μl DNA sample.
 +
*Mix by flicking the tubes and spinning for 15 seconds in a microfuge.
 +
*Denature in a heat block at 94 degrees centigrade for 3 minutes.
 +
*Microfuge for 15 seconds.
 +
*Add the following to the tubes.
 +
 +
{| border="3"
 +
! !! Test Sample 1(μl) !! Control Sample 2(μl)
 +
|-
 +
|  dNTP mix (2mM dATP, 2mM dGTP, 2mM dTTP, 0.5mM dCTP)|| 5||5
 +
|-
 +
| Cy3-dCTP (1mM)||3.75||-
 +
|-
 +
|Cy5-dCTP (1mM)||-||3.75
 +
|-
 +
|Klenow (BioPrime kit) ||1 ||1
 +
|}
 +
 +
*Mix by flicking the tube followed by a brief (<10 secs) spin a microfuge.
 +
*Incubate at 37 °C for 5 hours (the time of incubation determins the degree of amplification so you could vary it if you want or need to).
 +
*Use QIAquick PCR purification kit for cleaning up the labeld DNA. Elute DNA with two times 25μL of elution buffer from the column. The colour of the column after the wash step gives a first impression of the degree of labeling.
 +
*Measure the CyDye and DNA concentration of the samples at Nanodrop. DNA should be between 20 and 60ng when started with about 20ng immunoprecipitated DNA and CyDye between 2 and 6 (just to give an idea).
 +
 +
'''Hybridisation to microarray'''
 +
*The following protocol is thought for hybridisation of OGT arrays with Agilent SureHyb equipment. If you want to use other equipment adjustments to this protocol may be required.
 +
*Remove slide box from packaging and store slides until use in a dehumidified chamber. The slides should be stored in a light tight box.
 +
*When ready for use, remove slides from box. Wear clean powder free gloves at all times when handling the microarrays. Handling should be carried out in a low dust laboratory. Return unused slides to dehumidified chamber.
 +
*The arrays are printed on the same side of the slide to the label 'Agilent'.
 +
*Hybridisations are carried out in a 100μl volume per array.
 +
*The volume of CyDye labeld DNA must be reduced in a SpeedVac and be adjusted to 25μL.
 +
*Prepare the hybridisation buffer by pipetting the following into a tube. CARE Formamide is toxic.
 +
{| border="3"
 +
!  Component !! Volume for 125μL hybridisation (μL)!! Volume for one slide with four arrays
 +
|-
 +
|12xMES ||10 ||50
 +
|-
 +
|5M Sodium chloride ||24||120
 +
|-
 +
|Formamide ||24||120
 +
|-
 +
|0.5M EDTA ||5||25
 +
|-
 +
|10% Triton X100 ||12||60
 +
|}
 +
*The given volumes in the first column are for one hybridisation. If you have more than one you should do a master mix (second column) and use 75μL in the following step.
 +
*In a different tube join the differentialy labeled test and control DNA as follows:
 +
{| border="3"
 +
!  Component !! Volume for 125μL hybridisation (μL)
 +
|-
 +
|Cy3 labeld DNA||25
 +
|-
 +
|Cy5 labeld DNA||25
 +
|-
 +
|}
 +
*denature at 94°C for 3 min
 +
*Spin down and add sample mix in tube with hybridisation buffer (see above).
 +
*Place an Agilent SureHyb GASKET slide into an Agilent CHAMBER base.
 +
*Pipette 100μl of hybridisation mix onto the GASKET slide.
 +
*Place an OGT array slide onto the GASKET slide with the array side down (with 'Agilent' label) and in contact with the hybridisation mix.
 +
*Place the CLAMP ASSEMBLY on the slide and tighten the thumbscrew.
 +
*Some bubbles should form. These bubbles should be moving. If they are not, tap the chamber on the bench.
 +
*Hybridise at 55°C for 2 nights and one day (about 36 hours) in a light tight container, ideally in a hybridisation oven with a rotisserie. Fit the slides vertically and rotate the chambers at a speed at 4 rpm (setting 10 for Agilent hybridisation oven).
 +
 +
'''Washing and scanning of microarray'''
 +
*'''Note:''' Gloves should be changed after each wash step to not transfer CyDye.
 +
*Prepare the Wash solutions as follows
 +
**'''Wash 1 (1 litre)'''
 +
**20x SSPE 300ml
 +
**10% N-Lauroylsarcosine 0.5ml
 +
**Water 700ml
 +
Store at room temperature.
 +
*'''Wash 2 (1 litre)'''
 +
**20x SSPE 3ml
 +
**PEG200 1.8ml
 +
**Water 995ml
 +
**Store at room temperature.
 +
*Place 50ml of Wash 1 in a 50ml sterile tube.
 +
*Place 50ml of Wash 2 in a separate 50ml sterile tube.
 +
*Wearing gloves remove the slide from the hybridisation chamber with the GASKET slide still attached. CARE the hybridisation buffer contains formamide.
 +
*Place in a bath of Wash 1 and gently prise the GASKET slide from the OGT microarray under the surface of the Wash 1 buffer (use fingernails at the corners of the arrays).
 +
*Without the microarray drying out place the microarray into the 50ml tube with the Wash 1 buffer.
 +
*Rotate the tube on a rotary mixer at room temperature for 5 minutes.
 +
*Using clean forceps and without the microarray drying out, place the microarray into the 50ml tube with the Wash 2 buffer.
 +
*Rotate the tube on a rotary mixer at room temperature for exactly 5 minutes.
 +
*Using clean forceps remove the microarray and blow dry with dry nitrogen.
 +
*Insert the slide into the scanner and scan according the manufacturer’s instruction booklet. For Agilent scanner insert the slide into the Agilent slide holder. The array slide should be placed into the slide holder with the array side facing up. The 'Agilent' label should also be facing up. The non-labeled edge should be placed into the slide holder first. The slide should be scanned with the green laser (~532nm) and the red laser (~633nm).
 +
 +
'''Feature extraction with Agilent feature extraction software'''
 +
 +
*An XML file is supplied from OGT on the CDc to enable the data to be extracted using Agilent’s Feature extraction software. Please refer to the Agilent Feature extraction software documentation for full details.
 +
*Carry out Feature extraction as recommended by the software provider
 +
*A .txt file should be generated. When the .txt file is opened using Microsoft Excel, a spreadsheet should open that will contain one column with the genomic location of the probe on the array. There will be another column with the Green and Red signals (gProcessedSignal and rProcessedSignal).
 +
*One could than for example draw a graph of the genomic location on the X axis versus the ratio of the two dye signals (test/control).
 +
*For OGT array 010010, the genomic location of the probes on the file is based on the EMBL E.coli K12 MG1655 genomic sequence (ID U00096).
 +
*For OGT array  010011, the genomic location of the probes on the 0157 array is based on the EMBL E.coli 0157 genomic sequence (ID BA000007). The plasmid sequence used is AB011549.
 +
*There is also a ChIP browser availible for free download from OGT [http://www.ogt.co.uk/cms-analysis-software.htm] that helps to view ChIP data together with genomic location.
==Critical steps==
==Critical steps==
-
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
+
It is important to confirm that sonication results in fragments of a suitable size, i.e. <1 kb on average. This can be done by decrosslinking a sample of crosslinked cell extract, purifying by phenol extraction and ethanol precipitation, RNase treating and running on a gel.
==Troubleshooting==
==Troubleshooting==
Line 117: Line 345:
==References==
==References==
 +
 +
<biblio>
<biblio>
-
#Grainger2006NAR pmid=16963779
+
#Waldminghaus-2010 pmid=20602746
</biblio>
</biblio>
 +
 +
*Glycogen does not interfere with Klenow random labeling http://www.fermentas.com/catalog/reagents/glycogen.htm
==Specific Protocols==
==Specific Protocols==
Line 128: Line 360:
Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.
Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.
 +
 +
[[Category:Protocol]] [[Category:Escherichia coli]]

Current revision

back to protocols

This protocol is in developement and currently under investigation in the lab. Please contibute and watch changes in the next weeks!

Contents

Curators

Torsten Waldminghaus

Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently discussing the idea of protocol curators. Please contribute.

Abstract

ChIP-Chip stands for Chromatin Immunoprecipitation and chip in the sense of DNA microarray. It is a technique to determine the genome-wide binding sites of a DNA-binding protein. While the basic principle is the same for all species, there are some differences in handling cells. This protocol is developed and tested for E. coli. It should work the same way for other bacteria but that remains to be proven. Published protocols also exist for other bacterial species, including Bacillus subtilis, Caulobacter crescentus and Mycobacterium tuberculosis.

Because E. coli can be grown to high cell densities relative to eukaryotes, it is possible to generate sufficient DNA to label without using a PCR-based method. This method uses strand displacement primer extension with Klenow DNA polymerase and amplifies the DNA ~10-fold, while simultaneously incorporating dye-coupled nucleotide.

Related protocols can be found here:

Materials

  • 2 ml Ultralink protein A/G beads (catalog number 53132, Pierce)
  • Specific antibody for example RNA polymerase β subunit from Neoclone, Madison

Reagents

Material for about 50 chromatin immunoprecipitations:

  • 75 ml Formaldehyde (37%)
  • 500 ml 2.5 M Glycine
  • 1.5 l TBS (400 ml 5xTBS)
  • 50 ml TE
  • 20 mg/ml Proteinase K in TBS (store up to 1 year at -20 C)
  • 1 ml 100mM PMSF or 0.5 ml 250 mM Pefabloc


Final concentration For 50ml
Lysis Buffer
10mM Tris (pH 8.0)500μL 1M
20% sucrose10g
50mM NaCl500μL 5M
10mM EDTA1mL 0.5M
10 mg/ml lysozyme0.5g
  • Note: Prepare the 50ml without lysozyme and than add the corresponing ammount to 10 ml aliquotes and store them ad -20°C


Final concentration For 500ml
IP-Buffer (Immunoprecipitation Buffer)
50 mM HEPES-KOH pH 7.550ml 0.5M
150 mM NaCl15ml 5M
1 mM EDTA1ml 0.5M
1% Triton X 1005ml
0.1 % Sodium deoxycholate0.5g
0.1 % SDS5ml 10%


  • IP-Buffer with 500 mM NaCl: add 2.8mL 5M NaCl to 40mL IP-buffer


Final concentration For 50ml
Wash-Buffer
10mM Tris pH 8.0500µl 1M
250 mM LiCl5 ml 2.5M
1 mM EDTA100µl 0.5M
0.5% Nonidet-P40 (=Triton X114)250µl
0.5% Sodium deoxycholate2.5ml 10%


Final concentration For 10ml
Elution-Buffer
50 mM Tris (pH 7.5)500μL 1M
10 mM EDTA200μL 0.5M
1% SDS1mL 10%
ddH2O8.3mL


For labeling the DNA:

  • BioPrime kit Invitrogen (18094-011)[1]
  • Cy3-dCTP GE Healthcare (PA53021)[2]
  • Cy5-dCTP GE Healthcare (PA55021) [3]
  • Deoxynucleotides (2mM dATP, 2mM dGTP, 2mM dTTP, 0.5mM dCTP mix). Prepared as follows:
    • 4μl 100mM dATP GE Healthcare (27-2035-01)
    • 4μl 100mM dGTP
    • 4μl 100mM dTTP
    • 1μl 100mM dCTP
    • 187μl Water
    • Store at -20 °C
  • QIAquick PCR purification columns Qiagen (28104)[4]

Arrays

  • Oxfod Gene Technology (ogt) provides good arrays in 4*44k format compatibel with Agilent scanner and hybridisation equipment [5]
  • Hybridisation buffer materials:
  • 5M Sodium chloride Sigma (S6316)
  • 12x MES prepared as follows:
    • 3.52g MES free acid monohydrate Sigma (M2933)
    • 9.66g MES Sodium salt Sigma (M3058)
    • Water to 40ml
    • pH to 6.5 with 1M Hydrochloric acid
    • Add water to 50ml
    • Filter sterilise with a 0.45μm filter
    • Store at 4 °C
  • 10% Triton prepared as follows:
    • 1ml Triton X100 Sigma (T8787)
    • 9ml water
    • Store at room temperature
  • 100% Formamide Sigma (F9037-100ml)
  • 0.5M EDTA Sigma (E7889)
  • 20x SSPE Sigma (S2015)
  • 10% N-Lauroylsarcosine prepared as follows:
    • 1g N-Lauroylsarcosine Sodium salt Sigma (L9150-100g)
    • Water to 10ml
    • Store at room temperature
  • Polyethylene glycol (PEG) 200 Sigma (Fluka) (88440)

Equipment

  • DNA Microarray Hybridization Chamber - SureHyb from Agilent [6]
  • DNA Microarray Hybridization Oven from Agilent [7]
  • DNA Microarray Scanner from Agilent [8]

Procedure

This protocol has been broken up into 3 "days" but for E. coli it is possible to perform the entire ChIP experiment in a single day as well as the amplification/labeling step (which can be done overnight).


Formaldehyde cross link and sonication:

  • 50ml culture in LB or AB medium at 30 or 37 °C until OD600 0.5
  • Add 27μl formaldehyde (37%) per ml medium (substract what you took out for messuring OD) => final concentration of about 1%
  • Shake slowly (100 RPM) for 20 min at RT
  • Add 10 ml of 2.5 M glycine => final concentration of about 0.5 M
  • Keep shaking for 5 min
  • Harvest 10 ml of cells for each DNA-preparation (centrifuge 2500 g, 4°C, 10 min)
  • Wash twice in cold 10 ml TBS (20mM; see Material) pH7.5

Image:Pause point.png You can freeze the cell pellet and proceed later

  • Resuspend in 1 ml Lysis-Buffer
  • Incubate at 37 °C for 30 min (not shaking)
  • Add 4 ml of IP-Buffer
  • Add 50 μl of 100mM PMSF => final concentration of 1mM

Image:Critical step.png Sonicate so that DNA fragments are <1 kb on average (see "critical steps" section below). Using a Branson 450 sonifier with a microtip probe (inserted in the sample) 2 cycles of 30 s sonication are sufficient. Samples should be kept on ice at all times as they warm up considerably during sonication. It is important to leave the samples for at least 1 minute on ice between each round of sonication. Note that sonication parameters will vary according to the sonicator and probe used. It is possible to use a cup-horn sonicator but this requires much longer sonication and it is important to ensure that sonication is even for all samples being sonicated.

  • Centrifuge 12,000 g, 4 °C, 10 min


IP:

  • Use 800 μl aliquot for one immunoprecipitation experiment
  • Add 20 μl of a 50% slurry of protein A sepharose or protein A/G beads (note that the beads should be selected to work with the antibody being used)
  • Add specific antibody (for example 1 μl of RNA polymerase β subunit; see Material above)
  • Incubate at 4 °C overnight on a slow rotator or at room temperature for 90 minutes. Note that most antibodies work fine at room temperature but some require overnight incubation at 4 °C. This has to be determined empirically, as does the amount of antibody required for the IP.


  • Collect sephareose beads by centrifugation for 1 min at 3500 rpm
  • Pipett off supernatant and save as control DNA
  • All following wash steps should be on a rotator at room temperature for 3 min with 2 min centrifugation as above:
  1. Wash twice with 500 μl I-Buffer
  2. Wash with 500 μl I-Buffer with 500 mM NaCl
  3. Wash with 500 μl Wash-Buffer
  4. Wash with 500 μl TE
  • Add 100 μl of elution buffer. Gently pipet up and down two or three times in order to dislodge beads from the filter. Incubate 10 min in a 65 °C water bath. A water bath is used instead of other heating apparatuses in order to improve heat transfer.
  • Centrifuge beads 1 min at 3500 rpm, room temperature.Transfer supernatant into new 1.5 ml tube.
  • Add 80 μl TE and 1µl RNase A (20mg/ml) to the eluted DNA and to 100µl of the control DNA (see above)
  • Incubate for 90 min at 42°C
  • add 20 μl Proteinase K to each tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl). (Note: many protocols no longer have this step; I get equivalent data when I skip this)
  • To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. If not using Proteinase K, incubate overnight at 65 °C, or boil samples for 10 minutes.
  • Purify DNA by phenol extraction and ethanol precipitation or using a PCR purification kit (e.g. from Qiagen)
  • Elute or resuspend in 10-20 ul water
  • Measure DNA-content ideally at a NanoDrop (should be around 0.2 to 0.4 μg)
  • Use 1 and 10 ng DNA as template for quantitative PCR with primers that are specific for a known binding site of your DNA-binding protein and one negative control

Labeling for hybridisation to microarray:

  • The chromatin immunoprecipitated DNA samples should be in 20μl volume at a concentration of approximately 20ng/μl (more is also fine).
  • Note that one allways needs one test sample that will be labeld with one CyDye and a control labeld with another. As control one could use chromosomal DNA or DNA immunoprecipitated from a mutant strain of the DNA binding protein of choice or from different growth conditions etc.
  • add 20μL 2.5x Random primer (BioPrime kit) to each 20μl DNA sample.
  • Mix by flicking the tubes and spinning for 15 seconds in a microfuge.
  • Denature in a heat block at 94 degrees centigrade for 3 minutes.
  • Microfuge for 15 seconds.
  • Add the following to the tubes.
Test Sample 1(μl) Control Sample 2(μl)
dNTP mix (2mM dATP, 2mM dGTP, 2mM dTTP, 0.5mM dCTP) 55
Cy3-dCTP (1mM)3.75-
Cy5-dCTP (1mM)-3.75
Klenow (BioPrime kit) 1 1
  • Mix by flicking the tube followed by a brief (<10 secs) spin a microfuge.
  • Incubate at 37 °C for 5 hours (the time of incubation determins the degree of amplification so you could vary it if you want or need to).
  • Use QIAquick PCR purification kit for cleaning up the labeld DNA. Elute DNA with two times 25μL of elution buffer from the column. The colour of the column after the wash step gives a first impression of the degree of labeling.
  • Measure the CyDye and DNA concentration of the samples at Nanodrop. DNA should be between 20 and 60ng when started with about 20ng immunoprecipitated DNA and CyDye between 2 and 6 (just to give an idea).

Hybridisation to microarray

  • The following protocol is thought for hybridisation of OGT arrays with Agilent SureHyb equipment. If you want to use other equipment adjustments to this protocol may be required.
  • Remove slide box from packaging and store slides until use in a dehumidified chamber. The slides should be stored in a light tight box.
  • When ready for use, remove slides from box. Wear clean powder free gloves at all times when handling the microarrays. Handling should be carried out in a low dust laboratory. Return unused slides to dehumidified chamber.
  • The arrays are printed on the same side of the slide to the label 'Agilent'.
  • Hybridisations are carried out in a 100μl volume per array.
  • The volume of CyDye labeld DNA must be reduced in a SpeedVac and be adjusted to 25μL.
  • Prepare the hybridisation buffer by pipetting the following into a tube. CARE Formamide is toxic.
Component Volume for 125μL hybridisation (μL) Volume for one slide with four arrays
12xMES 10 50
5M Sodium chloride 24120
Formamide 24120
0.5M EDTA 525
10% Triton X100 1260
  • The given volumes in the first column are for one hybridisation. If you have more than one you should do a master mix (second column) and use 75μL in the following step.
  • In a different tube join the differentialy labeled test and control DNA as follows:
Component Volume for 125μL hybridisation (μL)
Cy3 labeld DNA25
Cy5 labeld DNA25
  • denature at 94°C for 3 min
  • Spin down and add sample mix in tube with hybridisation buffer (see above).
  • Place an Agilent SureHyb GASKET slide into an Agilent CHAMBER base.
  • Pipette 100μl of hybridisation mix onto the GASKET slide.
  • Place an OGT array slide onto the GASKET slide with the array side down (with 'Agilent' label) and in contact with the hybridisation mix.
  • Place the CLAMP ASSEMBLY on the slide and tighten the thumbscrew.
  • Some bubbles should form. These bubbles should be moving. If they are not, tap the chamber on the bench.
  • Hybridise at 55°C for 2 nights and one day (about 36 hours) in a light tight container, ideally in a hybridisation oven with a rotisserie. Fit the slides vertically and rotate the chambers at a speed at 4 rpm (setting 10 for Agilent hybridisation oven).

Washing and scanning of microarray

  • Note: Gloves should be changed after each wash step to not transfer CyDye.
  • Prepare the Wash solutions as follows
    • Wash 1 (1 litre)
    • 20x SSPE 300ml
    • 10% N-Lauroylsarcosine 0.5ml
    • Water 700ml

Store at room temperature.

  • Wash 2 (1 litre)
    • 20x SSPE 3ml
    • PEG200 1.8ml
    • Water 995ml
    • Store at room temperature.
  • Place 50ml of Wash 1 in a 50ml sterile tube.
  • Place 50ml of Wash 2 in a separate 50ml sterile tube.
  • Wearing gloves remove the slide from the hybridisation chamber with the GASKET slide still attached. CARE the hybridisation buffer contains formamide.
  • Place in a bath of Wash 1 and gently prise the GASKET slide from the OGT microarray under the surface of the Wash 1 buffer (use fingernails at the corners of the arrays).
  • Without the microarray drying out place the microarray into the 50ml tube with the Wash 1 buffer.
  • Rotate the tube on a rotary mixer at room temperature for 5 minutes.
  • Using clean forceps and without the microarray drying out, place the microarray into the 50ml tube with the Wash 2 buffer.
  • Rotate the tube on a rotary mixer at room temperature for exactly 5 minutes.
  • Using clean forceps remove the microarray and blow dry with dry nitrogen.
  • Insert the slide into the scanner and scan according the manufacturer’s instruction booklet. For Agilent scanner insert the slide into the Agilent slide holder. The array slide should be placed into the slide holder with the array side facing up. The 'Agilent' label should also be facing up. The non-labeled edge should be placed into the slide holder first. The slide should be scanned with the green laser (~532nm) and the red laser (~633nm).

Feature extraction with Agilent feature extraction software

  • An XML file is supplied from OGT on the CDc to enable the data to be extracted using Agilent’s Feature extraction software. Please refer to the Agilent Feature extraction software documentation for full details.
  • Carry out Feature extraction as recommended by the software provider
  • A .txt file should be generated. When the .txt file is opened using Microsoft Excel, a spreadsheet should open that will contain one column with the genomic location of the probe on the array. There will be another column with the Green and Red signals (gProcessedSignal and rProcessedSignal).
  • One could than for example draw a graph of the genomic location on the X axis versus the ratio of the two dye signals (test/control).
  • For OGT array 010010, the genomic location of the probes on the file is based on the EMBL E.coli K12 MG1655 genomic sequence (ID U00096).
  • For OGT array 010011, the genomic location of the probes on the 0157 array is based on the EMBL E.coli 0157 genomic sequence (ID BA000007). The plasmid sequence used is AB011549.
  • There is also a ChIP browser availible for free download from OGT [9] that helps to view ChIP data together with genomic location.

Critical steps

It is important to confirm that sonication results in fragments of a suitable size, i.e. <1 kb on average. This can be done by decrosslinking a sample of crosslinked cell extract, purifying by phenol extraction and ethanol precipitation, RNase treating and running on a gel.

Troubleshooting

Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.

Notes

Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.

It might also be good to add an image to show the workflow and timescales for experiment planning.

Acknowledgments

Acnkowledge any help you had in development, testing, writing this protocol.

References

  1. Waldminghaus T and Skarstad K. . pmid:20602746. PubMed HubMed [Waldminghaus-2010]

Specific Protocols

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