Permanganate genomic footprinting Drosophila cells
(written 1/22/07, developed by Ruopeng Fan).

Ruopeng's method is designed for cells that grow attached to the dish.  Ruopeng determined that 1 minute treatment with 10 mM KMnO4 on ice works well.  Longer treatments or higher concentrations results in oxidation of thymines that don't correspond to known positions of Pol II.


Cells.

Cells should be growing exponentially and should look healthy in the microscope.  Their density should about 5 million cells per ml.  Put 2 million cells in 2 ml media in a 35mm cell culture dish.  Leave the cells overnight.

Permanganate treatment of cells.
Prepare fresh solutions of 10 mM  KMnO4 and 5X KMnO4 Stop solution. For a 35 mm dish of cells, you'll need 800 ul of 10 mM KMnO4 and 200 ul of 5X KMnO4 Stop solution.  At some point, you'll need to prepare DNA from cells that have not been treated with permanganate to serve as a negative control and as a source for treating purified DNA with permanganate.  This can be done in parallel with the permanganate treatment of cells by using PBS in place of 10 mM KMnO4.
10 mM KMnO4 - store on ice after the permanganate has dissolved

5 ml PBS

7.9 mg KMnO4

Rock at room temp for 15' and store on ice.  It is very important to thoroughly dissolve the permanganate. The dark color of the solution makes it difficult to see when the crystals have dissolved.

 5X MnO4 stop solution - store at room temp. so the SDS doesn't precipitate

50 ul 1 M Tris-Cl pH 7.5 

10 ul 5 M NaCl 

100 ul 0.5 M Na2EDTA pH 8 

250 ul 10% SDS 

70 ul ß-MeOH

520 ul H2O

(If you want to heat shock the cells in a 35mm dish, do the following. Prewarm 1 ml of media without FBS to 37oC.  Pipet away the 2 ml media in the dish and add 1 ml of prewarmed media.  Incubate the cells for 15 minutes at 37oC.  Carefully pipet away the warm media and proceed with permanganate treatment.)

For a 35 mm dish of cells, pipette away the culture media and immediately add 800 ul of cold permanganate solution.  Place the dish on ice for 60 seconds and agitate constantly so the cells are evenly treated.  Remove the dish from the ice and add 200 ul of 5X KMnO4 Stop solution.  Gently swirl the dish until the purple color has dissappeared.  Add 50 ug of proteinase K and mix.  Allow the dish to stand at room temperature for 20 minutes.  Then gently triterate the solution and transfer it to a microfuge tube.  Incubate the sample for at least 2 hours at 37oC.   

Divide that sample in half and extract each half with a sequence of equilibrated phenol (pH 8), phenol/chloroform/isoamylalcohol (25:24:1), and chloroform. Each extraction is done with 500 ul of the organic solution. Samples are shaken for 5 minutes and centrifuged for 5 minutes to separate phases (room temperature). For both of the phenol extractions, the aqueous phase is transferred to a fresh tube. For the chloroform extraction, the chloroform layer is removed by pipetting it out from underneath the aqueous phase.

Each half of the sample will be approximately 500 ul.  To each half, add 50 ul of 3M Na Acetate pH 7 and 1 ml of cold ethanol.  Thoroughly mix by inverting the tube and incubate at room temperature for 10 minutes.  It is important not to incubate the sample on ice because this seems to precipitate something that interferes with the subsequent LM-PCR reactions. 

Microfuge at room temperature for 20 minutes.  Discard the supernatant and rinse the pellet with cold 75% Ethanol.  Microfuge in the cold for 5 minutes and discard the supernatant. Allow the samples to air-dry.

Dissolve the nucleic acid pellet in 50 ul of TE (10 mM Tris pH 7.5, 1 mM EDTA).

Determine the concentration of DNA by measuring 1 ul of the sample on the Fluorometer, which does not detect contaminating RNA.  

Store samples in the refrigerator for short term or in the freezer for long term.


Permanganate treatment of naked DNA.

In order to interpret the pattern of permanganate reactivity that occurs in vivo, the pattern must be compared to that which occurs in naked (protein-free) genomic DNA.

Put 1 ug of DNA from cells and dissolved in 10 ul of TE into a microfuge tube.  Add 100 ul of cold 10 mM permanganate (dissolved in PBS) and incubate on ice for 60 seconds.  Stop the reaction with 30 ul of KMnO4 Stop solution. 

Ethanol precipitate the DNA by adding 14 ul of 3M Na acetate pH 7 and 350 ul of Ethanol, mixing thoroughly and incubating on ice for 15'.  Microfuge in the cold for 20', discard the supernatant and wash pellet with cold 75% ethanol. Note that this DNA is not phenol extracted since it should already be protein-free.

Air dry the DNA and then dissolve it in 50 ul of TE. Quantify the DNA with a fluorometer.


Piperidine cleavage of the permanganate-treated DNA.

Piperidine cleaves the DNA at the oxidized thymine residues. One hundred nanograms of DNA should start in a total volume of 15 ul of TE. The DNA is put in a siliconized 0.65 ml tubes. Add 75 ul of H2O and 10 ul of piperidine. Heat the samples at 90°C for 30 minutes in the thermocycler.

After 30 minutes, add 300 ul of water to the DNA solution and transfer each sample to a 1.7 ml tube.  Isobutanol extract 2 times using 800 ul each time and 1 time using 400 ul of isobutanol.  Isobutanol will be the top phase and should be discarded.  Extract one time with ether.  Adjust the volume of DNA to 100 ul by adding water.  Ethanol precipitate the DNA using 10 ul of 3M Na acetate pH 7.0 and 250 ul of ethanol.  Precipitate the DNA and wash with 75% ethanol in the routine way.  Dissolve permanganate samples in 10 ul of 1/2 X TE.   Transfer the DNA to a fresh siliconized 600ul tube. This transfer is essential since there is something about the tube where the piperidine reaction was done that completely inhibits the Vent polymerase. Its now ready for LM-PCR.


Generation of G/A markers using depurination with formic acid.

Formic acid depurinates the DNA and subsequent cooking with piperidine cleaves the DNA backbone. Formic acid treating 1 ug of DNA should yeild enough material for 10 gels.

In a siliconized 1.7 ml tube, combine 1 ug of genomic DNA in 10 ul of TE with 10 ul of H2O and 50 ul of 99% formic acid. Incubate at 15°C for 5'.

Add 200 ul of cold 0.3 M Na acetate pH 7.0, 50 ug/ml tRNA to stop depurination. Add 750 ul of cold Ethanol and ice for 15 minutes. Microfuge in the cold for 15 minutes to collect the DNA. Wash pellet with 200 ul of 75% cold Ethanol and air dry.

Dissolve the DNA in 90 ul of H2O and transfer to a siliconized 600 ul tube. Add 10 ul of piperidine and incubate at 90°C for 30 minutes.

Remove the piperidine as described above for piperidine cleavage of permanganate-treated DNA.  Air dry the final ethanol precipitate and dissolve in 50 ul of 1/2X TE.

By subjecting 10 ul of the DNA to the LM-PCR protocol, enough radiolabelled DNA will be made to easily detect a G/A ladder on at least two gels with overnight exposures.


LM-PCR analysis. (Note that an updated version of this protocol presented in the form of a worksheet by going here - there are some volume changes to streamline the protocol.)

The protocol is derived from Garrity and Wold's procedure found in Current Protocols. Slight modifications were introduced by Jan Weber, and the current description is specific for the set of primers that anneal to ß-gal sequences downstream from the transformed promoters. These primers are called TR-1, TR-2, and TR-3. Obviously, if you intend to use a different set of primers, you will probably have to adjust the PCR conditions. Some examples can be found here.

Be sure to mix all solutions thoroughly using gentle triteration. To improve accuracy, prepare master mixes of various solutions. Prepare the master mixes by multiplying the amount of material for one reaction by the sum of 0.5 and the number of samples being processed. This ensures that there will be enough master mix for all of the samples. Also, check the PCR programs to make sure they are correct - sometimes they get modified and not returned to their original state.

Combine 10 ul of DNA (100 ng in TE) with 20 ul of First strand synthesis mix.  In the thermocycler, incubate at 95°C for 5 minutes, 50°C for 30 minutes, and 76°C for 10 minutes. Spin down condensate and place on ice.

First strand synthesis mix recipe (20ul/rxn)

12.75 ul H2O 

6ul 5X First Strand Buffer* (heat briefly to 37oC to ensure that the geletin is fully dissolved)

0.5 ul of 1 pmoles/ul TR-1

0.5 ul 10 mM dNTPs 

0.25 ul 2U/ul Vent polymerase (NEB #254L)

Shortly before use, premix  17.5 ul of Modified Ligation Dilution Solution (MLDS)* and 2.5 ul of 1 mg/ml BSA per  sample; then add this mix to the sample. 

Next, add 25 ul of Ligation Mix. Incubate overnight at 15°C.
Ligation Mix recipe (25ul/rxn)

9.25 ul Modified Ligation Premix*

7.5 ul 10 mM rATP 

1.25 ul 1mg/ml BSA 

5 ul Linker pair (A':B at 20 pmoles/ul) 

2 ul T4 DNA ligase (1Weiss Unit/ul)
*11/13/96: I reduced the ligase from 3U (original protocol) to 2U and added 1ul of water to make-up the difference. Strong signals were still obtained. Add 9.4 ul of salt precipitation mix* and 220 ul cold Ethanol. Mix well and place on ice for at least 15 minutes. Microfuge in cold for 20 minutes. Wash one time with 100 ul of 75% ethanol, microfuging to secure the precipitate. Air dry the pellet.
Dissolve the DNA precipitates in 30 ul of H2O.  Place samples on ice.  Add 70 ul of Vent amplification buffer.
Vent Amplification buffer recipe (70ul/rxn)

20 ul 5X Vent Amplification Buffer* (heat briefly to 37oC to ensure that the geletin is fully dissolved)

1 ul of 10 pmole/ul TR-2 

1 ul of 10 pmole/ul Linker primer A' 

2 ul 10 mM dNTP 

0.5 ul Vent polymerase 

45.5 ul H2O

Perform 18 amplification cycles. (This can be altered if your signals are weak.  I have gone up to 20 cycles.) The first cycle should have a 3 minute denaturing step at 95°C and the remaining should be for 1 minute. All cycles have annealling steps of 2 minutes. For TR-2, this is 58°C. The first 9 cycles have extension steps of 3 minutes and the last 9 cycles have extension steps of 4 minutes. All extension steps are at 76°C. The two sets of 9 cycles is provided by program 2. Following amplification, the tubes are placed on ice.

If it is not already done, radiolabel TR-3 with kinase. The following reaction will give you enough primer to label 20 samples if you adjust the final volume of the primer so that it is a little more than 100 ul. Combine 6.3 ul of H2O, 1 ul of 10X kinase buffer, 0.4 ul of 0.1 mM TR-3, 2 ul 32P-g ATP, and 0.3 ul kinase. Incubate at 37°C for 30 minutes. We used to purify the oligonucleotide on a Nensorb column. As of September 1997, we have been purifying radiolabelled oligonucleotides on Stratagene's NucTrap columns. Since the oligonucleotide is recovered in a aqueous solution (rather than 50% methanol) it can be used straight from the NucTrap column. You should obtain at least 106 cpm/pmole. As of 2005, we have been purifying radiolabelled oligonucleotides with spin columns from eith BioRad or Amersham.

After the 18 cycles of amplification, proceed with the labelling reaction. Add 20 ul of labelling mix to each sample. Perform two amplification cycles using program 3. The first cycle is initiated with a 3 minute denaturation at 95°C and the second cycle is for 1 minute. Annealling is done for 2 minutes at 62°C. Extensions are done for 10 minutes at 76°C. Following the labelling cycles, hold tubes at room temperature.

Labelling mix recipe (20ul/rxn *)

9.5 ul H2O 

4 ul 5X Vent Amplification Buffer* (see above) 

1 ul 10 mM dNTP 

5 ul radiolabelled TR-3 (approximately 2 pmoles) 

0.5 ul Vent Polymerase
*11/13/96: In my last experiment, I realized that I was going to run out of mix. I added 10ul rather than 20ul to the last two samples and then dissolved the resulting samples in 6ul rather than 12ul of sequencing loading buffer. The samples were well matched on the gel indicating that reducing the labelling mix by 2 results a proportional decrease in labelled material. There was still plenty for the gel.

At the end of the labelling reaction, add 14 ul of Stop solution*.  Transfer 120 ul of each reaction to a fresh 1.7 ml tube (the larger tube facilitates the organic extraction).  Extract one time with 100 ul of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) {Caution, highly radioactive!). Transfer 100 ul of the aqueous phase to a fresh 1.7 ml tube and add 250 ul of cold Ethanol. Chill on ice for 15 minutes and then microfuge for 15 minutes. Remove supernatant and wash pellet with 100 ul of 75% ethanol, microfuge and discard supernatant. Air dry the samples.

Dissolve precipitates in 10 to 15 ul of sequencing loading buffer. For gel analysis, boil 3-5 ul and reserve the rest for a subsequent gel if this is needed.


Recipe for 8% sequencing gel mix (the following is enough for 5 gels @ 30ml/gel).  This stock is stored at room temperature and is good for only a few weeks.

Dissolve the following in water to give a total volume of 157.5 ml:

84 g Urea

13.3 g  acrylamide

0.7 g  bis-acrylamide
Add 17.5 ml of 10X TBE. This results in a final volume of 175 ml of 8% gel mix.

For our sequencing plates, mix 35 ml of sequencing gel mix, 200 ul of 10% ammonium persulfate, and 23 ul of TEMED.  Allow the gel to polymerize for at least 1 hour. 
LMPCR Stock solutions:
5X First Strand Buffer (Store in freezer, warm to 37oC before use to thoroughly dissolve gelatin) Modified Ligation Dilution Solution (MLDS, store in freezer) Modified Ligation Premix (Store in freezer) Salt Precipitation Mix (Store in freezer) 5X Vent Amplification Buffer (Store in freezer, warm to 37oC before use to thoroughly dissolve gelatin) Stop Solution (Store in freezer) Preparation of A':B linker pair
In a 1.5 ml microfuge tube, prepare 1 ml of solution containing final concentrations of 250 mM Tris-Cl pH 7.7, 20 uM Linker A' and 20 uM Linker B.  Heat the solution to 95oC for 5 minutes and then float the tube in a beaker containing 250 ml of water adjusted to 70oC.  Allow the water to cool to room temperature and then leave the tubes at room temperature for at least one more hour.  The beaker of water containing the tube of oligonucleotides is then place on ice so that it slowly cools to 4oC and then the tubes are left on ice overnight.  Store the oligonucleotide solution at -20oC.  Don't warm the solution above room temperature when you thaw the tubes.

Alternatively, the oligonucleotides can be annealed in the PCR machine.  Divide the solution above into 5 x 200 ul portions in PCR tubes.  Subject to the following: 95oC for 5', 70oC for 5', 65oC for 5' , continue decreasing 5oC increments and incubating 5' until the samples reach 20oC, 20oC for 60', 18oC for 5', 16oC for 5', 14oC for 5', 12oC for 5', 10oC until its convenient to put at 4oC overnight.  Finally, store in the freezer.