ChIP-Chip E. coli-PDF

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.

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

Lysis Buffer

• Note: Prepare the 50ml without lysozyme and than add the corresponing ammount to 10 ml aliquotes and store them ad -20°C

IP-Buffer (Immunoprecipitation Buffer)

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

Wash-Buffer

Elution-Buffer

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 0.2

• 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 50 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

You can freeze the cell pellet and proceed later

• Resuspend in 300 μl IP-Buffer with 1mM Pefabloc

Sonicate so that DNA fragments are on average about 500 bps (see “critical steps” section below). With a Bioruptor from Diagenode use 48 cycles of 30 s sonication and 30 s cooling. 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.

• Prepare two 2ml tubes with 1.5ml IP-Buffer each.

• Add 150 µl of sonicated extract to each tube with IP-Buffer

• Centrifuge 12,000 g, 4 °C, 10 min

IP:

• Transfer 800 μl aliquots of the supernatant in 4 tubes for immunoprecipitation

• 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 700 μl I-Buffer
2. Wash with 700 μl I-Buffer with 500 mM NaCl
3. Wash with 700 μl Wash-Buffer
4. Wash with 700 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 of all 4 tubes into 1 new 1.5 ml tube.

• Centrifuge tube for 1 min at 3500 rpm, room temperature to pellet remaining agarose beads.

• Transfer supernatant into new 1.5 ml tube.

• Add 340 μl TE and 4µl RNase A (20mg/ml)

• 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 4×100μl). (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

Isolation of control DNA:

• control DNA is isolated from the supernatant of the IP reaction (see above)

• Incubate 500µl of IP supernatant at 95°C for 10 min

• Add 2 µl RNase A and incubate in waterbath at 42°C for 90 min

• Extract DNA with 1 x phenole and 2 x chlorophorm

• Precipitate with 1 ml ethanol and 40 μL 3M Na-Acetat and 1.5µl Glyco-Blue over night at -20°C

• spin down DNA at 4°C for 15 min at max. speed, wash in 70% ethanol and resuspend pellet in 30 μL dH2O

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.

• 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.

• 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:

• 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.

Acknowledgments

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

References

1. Waldminghaus T and Skarstad K. ChIP on Chip: surprising results are often artifacts. BMC Genomics. 2010 Jul 5;11:414. DOI:10.1186/1471-2164-11-414 | PubMed ID:20602746 | HubMed [Waldminghaus-2010]

• Glycogen does not interfere with Klenow random labeling http://www.fermentas.com/catalog/reagents/glycogen.htm