Has anyone tried to sonicate Jurkat cells for ChIP assay? - (Oct/26/2005 )

Hi,

I'm having problems with sonication for Jurkat cells. I did try couple methods like, MNase treated (I did titration from 25U to 200U in total cells from 5x10^7 to 2x10^8 cells) for 10minutes in 37C in water bath, then sonicate (titration again) at 60% duty, 50% amplitude, 1pulse = 1minute of 60% duty ( for each sec: 0.6 sec on, 0.4 sec off) with Branson Sonifier 450D and double step micro tip. Finally, I found out that with at least 100U MNase treatment and 15 pulses of sonication. I obtained smear from 500-1.5kb. However, I saw ladder bands in the smear (due to MNase). Then I used RNA Pol II from Active Motif to IP. Sequenase with randomers with specific tail then PCR with specific primer of the IP and no ab control. I got very good yield around 10ug with 30 cylces. However, I used this PCR product and tested them with GAPDH primers to see fold changes. I didn't see any (use 10ng PCR template per rxn) with SYBR PCR neither. I did fragment the samples then label them at 3' end (TDT) and hybridize onto Affymetrix promoter arrays and tiling arrays ( human sets). I got through all analyze tools that bioinformatic experts designed and IGB to find out if my samples showed difference between IP and no ab control. Unfortunately, I didn't see that. What happened here? Is this due to the randomers in sequenase? or clean up step after sequenase step prior PCR? I did check my GAPDH primers with Input--genomic DNA (titrate from 0.2ng to 10ng per rxn); they were working fine (specific band showed up around 160bp).

I did try sonicating cells without MNase, but I got smear above 4kb (too big!) with 15 pulses and 20 pulses; so with 5 pulses different I didn't see any change in the fragmentation. I'm afraid that too many pulses could damaging fixing (proteins-DNA binding).
Hope someone has been through these problems, and has resolved and trouble-shot them.

Thanks very much--Vin

hi vin,

for sonication conditions, you may want to take a look at
sonication and other woes topic.

To get efficient sonication you need to ensure your chromatin solution is not foaming, if it is, this will reduce the sonication efficiency. You can try to reduce the duty cycle and amplitude to get smaller fragments.

I am not too sure what you are trying to find.......i take it you want to see what parts of the genome are actively transcribed in Jurkat cells with an RNA Pol IP? GAPDH is a positive IP control and will be highly abundant in both your input and IP.

if enrichment of chromatin with RNA pol II bound is all you are looking for and you are not seeing this it may be that your IP has failed. The antibody is not binding to the chromatin efficently. This can be caused by a number of things such as, a poor antibody (this can be tested by western blotting) or that the epitope is masked in your chromatin by overfixation.

You may need to titrate your fixation times to optimise your antibody actually binding to the fixed chromatin.

Good luck!

Nick

Hi, I am currently performing native ChIP on Jurkat cells using just MNase and no sonication. I've found that the key is to do trial digests of your nuclei each time to get the icubation time just right for each cell line and aliquot of MNase.
I normally use 10U of MNase/0.1mg of nuclei in a 0.2ml volume for 3, 5, 7 ,9 and 11 minutes and check the DNA from these as well as the undigested nuclei.
It adds a bit of time to the process but as well getting the conditions right for the main digest, it also tells you if the undigested nuclei have started to degrade.... Never a good thing.

Anton

Hi Nick,

I just want to make sure ChIP assay working; so I just try out with RNA Pol 2, which expresses GAPDH. I want to have a result to show me that the assay is working by expression fold-changes in IP and NAC (no ab control). There was no foaming and sonication has been tested and optimized with genomic DNA and different cell lines (HL60). I just afraid that my method of dU ratio incorporation in PCR then using APE1 and UDG enzymes to fragment creating big fragment (200bp), which's supposed to have around 50bp. Another point that I'm worried is that GC content of promoter regions in the promoter arrays don't have much T's; so dU's don't incorporate. I have to try to use regular dNTPS then DNase fragmentation to see any change in array results.
About antibody (pol2), I have run expt in HL60 cells; it shows working. After purifing reverse cross-linked IP and NAC with Qiagen columns (cDNA) did run regular PCR with GAPDH and SYBR; there are 7-8 cts different between IP and NAC. However, I use sequenase-->PCR product to redo GAPDH PCR (start out with 10ng) and SYBR; I don't see the maintainance of cts between IP and NAC!?
Any suggestions or comments?

Thanks--Vin

Hi Raffy,

Sorry for late response since I just got back from a month vacation in southeast asia.
The protocol for MNase digestion is simple by following these steps:
1-Wash the pellet 3 times with 10 ml run-on lysis buffer; spin at 1000 rpm table top
Run-on Lysis Buffer:
10 mM Tris-Cl pH 7.5
10 mM NaCl
3 mM MgCl2
0.5% Igepal-CA630 (from Sigma)
1 mM PMSF ( add fresh)
2.-Resuspend pellet in 1.5 ml MNase buffer
MNase Reaction Buffer:
10 mM Tris-Cl pH 7.5
10 mM NaCl
3 mM MgCl2
1 mM CaCl2
4% Igepal-CA630
1 mM PMSF (add fresh)
3.-Add:
RNase (10 mg/ml; Sigma R-5125; remove DNAase prior to using) to final concentration of 100 g/ml
100 U MNase (USB cat. # 70196Y; dissolve to desired concentration in sterile 50% glycerol) [usually do two conc. For ex. 100 U and 200 U]
4.-Incubate at 37 oC, 10 min, rotating wheel
5.-Add 30 ul 200 mM EGTA to stop the reaction
6.-Check fragmentation efficiency by decross-link with Proteinase K (2ul of 20mg/mL per rxn at 65C for 6-overnight)

good luck, and let me know if it works out for you--Vince

Hi chipvince

Did you check next? If you did, could you say how different it was?

I just afraid that my method of dU ratio incorporation in PCR then using APE1 and UDG enzymes to fragment creating big fragment (200bp), which's supposed to have around 50bp. Another point that I'm worried is that GC content of promoter regions in the promoter arrays don't have much T's; so dU's don't incorporate. I have to try to use regular dNTPS then DNase fragmentation to see any change in array results.

Thank you so much.

hi chipvince
i am trying to do chip on chip assay where using random primer i am amplifying immunoprecipitated dna using sequenase ....but iam not getting any amplification .....can you suggest me something