ChIP-Chip E. coli

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*Mix by flicking the tube followed by a brief (<10 secs) spin a microfuge.
*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).
*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).
* Amplification for microarrays is done using the BioPrime kit (Invitrogen) with dye-coupled dCTP (Cy3 or Cy5 dye), according to the manufacturer's instructions. Typically 5-6 individual ChIP samples are combined for a single amplification/labeling reaction
* Amplification for microarrays is done using the BioPrime kit (Invitrogen) with dye-coupled dCTP (Cy3 or Cy5 dye), according to the manufacturer's instructions. Typically 5-6 individual ChIP samples are combined for a single amplification/labeling reaction

Revision as of 04:19, 31 July 2008

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 06:29, 17 March 2008 (CDT)

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 [1]. This method uses strand displacement primer extension with Klenow DNA polymerase and amplifies the DNA ~10-fold, while simultaneously incorporating dye-coupled nucleotide.

Other ChIP-Chip 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
  • 50 ml Lysis Buffer:
    • 10mM Tris (pH 8.0)
    • 20% sucrose
    • 50mM NaCl
    • 10mM EDTA
    • 10 mg/ml lysozyme
  • 500 ml IP-Buffer (Immunoprecipitation Buffer):
    • 50 mM HEPES-KOH pH 7.5
    • 150 mM NaCl
    • 1 mM EDTA
    • 1% Triton X 100
    • 0.1 % Sodium deoxycholate
    • 0.1 % SDS
  • 30 ml IP-Buffer + 500 mM NaCl
  • 30 ml Wash-Buffer:
    • 10mM Tris pH 8.0
    • 250 mM LiCl
    • 1 mM EDTA
    • 0.5% Nonidet-P40
    • 0.5% Sodium deoxycholate
  • 10 ml Elution-Buffer:
    • 50 mM Tris (pH 7.5)
    • 10 mM EDTA
    • 1% SDS

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:
    • 17.6g MES free acid monohydrate Sigma (M2933)
    • 48.3g MES Sodium salt Sigma (M3058)
    • Water to 200ml
    • pH to 6.5 with 1M Hydrochloric acid
    • Add water to 250ml
    • Filter sterilise with a 0.45μm filter
    • Store at 4 °C
  • 10% Triton prepared as follows:
    • 10ml Triton X100 Sigma (T8787)
    • 90ml water
    • Store at room temperature
  • 100% Formamide Sigma (F9037-100ml)
  • 0.5M EDTA Sigma (E7889)
  • 20x SSPE Sigma (S2015)
  • 20% N-Lauroylsarcosine prepared as follows:
    • 20g N-Lauroylsarcosine Sodium salt Sigma L9150-100g
    • Water to 100ml
    • Store at room temperature
    • Polyethylene glycol (PEG) 200 Sigma (Fluka) 88440


Equipment

Any equipment used to perform the protocol (link to a method for using them).

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).

1. Day

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.


2. Day

  • Collect each sample on Spin-X column by centrifugation for 1 min at 3500 rpm
  • All following wash steps should be on a rotator at room temperature for 3min 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
  • Place filter unit containing the beads into a new 1.5-ml microcentrifuge tube (dolphin-nosed tubes are best because they accommodate the Spin-X column more easily) 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.
  • Centrifuge beads 1 min at 3500 rpm, room temperature. Discard filter with beads. Transfer the eluate into new 1.5 ml tube.
  • Add 80 μl TE and 20 μl Proteinase K to the 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.


3. Day

  • 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).
  • Amplification for microarrays is done using the BioPrime kit (Invitrogen) with dye-coupled dCTP (Cy3 or Cy5 dye), according to the manufacturer's instructions. Typically 5-6 individual ChIP samples are combined for a single amplification/labeling reaction


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. Grainger DC, Hurd D, Harrison M, Holdstock J, and Busby SJ. . pmid:16301522. PubMed HubMed [Grainger-2005]
  2. Grainger DC, Hurd D, Goldberg MD, and Busby SJ. . pmid:16963779. PubMed HubMed [Grainger2006NAR]
All Medline abstracts: PubMed HubMed

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