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ChIP- reproducibility with sonicated DNA size range on agarose gel?
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+ Agarose gel
+ Chip-sequencing
+ Chip-sequencing analysis
+ Chromatin immunoprecipitation (chip)
+ Jurkat cells
+ Ripa buffer
Posted by
Abrar Hafiz
ChIP- reproducibility with sonicated DNA size range on agarose gel?
Hi Ghina, Thanks for the NaCl tip. That does wonder to the reverse crosslinking process. Now I am able to get the desired fragment length but now my ChIP-qPCR is not working. Would you mind sharing your entire ChIP protocol with me?
Hi Ghina, Thanks for the NaCl tip. That does wonder to the reverse crosslinking process. Now I am able to get the desired fragment length but now my ChIP-qPCR is not working. Would you mind sharing your entire ChIP protocol with me?
Hi Saptha, I finally solved my issues it was the reversed cross-link, the DNA extraction quality and also the amount of EtBr in my gel. I now treat 1h with Rnase A at 37°C, reverse the cross-link ON 65°C with 0,2M NaCl (NaCl really changed the profile! ) then treat 1h with PK at 45°C, I am using ActivMotif kit to extract DNA for qPCR (with the Nanodrop I can see i have no phenol/Chlo contamination my DNA is clean) and now i can see my samples even with only 500ng on my gel also I now add more EtBr on my gel (10µL for like 150mL) . My fragments are about 200-400bp I use JLat cells (jurkat) I sonicate 10M cells (Diagenode kit medium SDS) , 7 cycles and I only fix 10'. Analysing the fragments size seems more complicated than the ChIp itself !! Hope this can help you out ! https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2077865/pdf/1746-4811-3-11.pdf I think more than 10 -15 cycles is way too much for jurkat cells probably like me ,you also have an issue after sonication : reverse cross link dna isolation , gel migration?! try to run sonicated and non sonicated samples on your gel +/- NaCl +/- PK and try a different DNA extraction technique 1µg-500ng should be ok try not to load too much on your gel ! Good luck!!
Hi Saptha, I finally solved my issues it was the reversed cross-link, the DNA extraction quality and also the amount of EtBr in my gel. I now treat 1h with Rnase A at 37°C, reverse the cross-link ON 65°C with 0,2M NaCl (NaCl really changed the profile! ) then treat 1h with PK at 45°C, I am using ActivMotif kit to extract DNA for qPCR (with the Nanodrop I can see i have no phenol/Chlo contamination my DNA is clean) and now i can see my samples even with only 500ng on my gel also I now add more EtBr on my gel (10µL for like 150mL) . My fragments are about 200-400bp I use JLat cells (jurkat) I sonicate 10M cells (Diagenode kit medium SDS) , 7 cycles and I only fix 10'. Analysing the fragments size seems more complicated than the ChIp itself !! Hope this can help you out ! https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2077865/pdf/1746-4811-3-11.pdf I think more than 10 -15 cycles is way too much for jurkat cells probably like me ,you also have an issue after sonication : reverse cross link dna isolation , gel migration?! try to run sonicated and non sonicated samples on your gel +/- NaCl +/- PK and try a different DNA extraction technique 1µg-500ng should be ok try not to load too much on your gel ! Good luck!!
Hi Saptha ! As I said in my first comment I am very new at that ChIp thing, all I know I get it from literature commercial protocols home made peotocols and through research gate questions haha! My target proteins are histones that is why I can fix my cells only 10min from what I read when trying to solve my issues when you work on transcription factors or factors that do not directly bind on your dna you have to fix longer .. just as you do. I really don't know if this will impact sonication drasticallyI think you can find an answer for that in diagenode protocol at the very end of the booklet. I sonnicate mine 7cycles 30sOn/30sOff I tried 9 don't see big difference and 10 is too much for me 100-300bp NaCl really helps with reversing cross-link and if you fixed you cells 15min you will defently need NaCl .. I read some people needed to decross link for almost 24h when they fixed too long (the internet is full of ChIp-desperate students like us haha). About the 3kb fragment have you checked for contamination in your cells? Do you use salmon sperm DNA somewhere in your protocol for instance as a carrier? Or to avoid non specific binding during you chIP? Also ising the extraction kit helps gain time and procede to several tests. If I can give you an advice if you want to solve your issue : fresh start put one standard protocol (you can find a paper that focus on transcription factors and check their protocol) than start changing one parameter at a time! Also compare the different old protocols you used to gain time. Good Luck be patient and stubborn like your 3kb fragment!! ;)
Hi Saptha ! As I said in my first comment I am very new at that ChIp thing, all I know I get it from literature commercial protocols home made peotocols and through research gate questions haha! My target proteins are histones that is why I can fix my cells only 10min from what I read when trying to solve my issues when you work on transcription factors or factors that do not directly bind on your dna you have to fix longer .. just as you do. I really don't know if this will impact sonication drasticallyI think you can find an answer for that in diagenode protocol at the very end of the booklet. I sonnicate mine 7cycles 30sOn/30sOff I tried 9 don't see big difference and 10 is too much for me 100-300bp NaCl really helps with reversing cross-link and if you fixed you cells 15min you will defently need NaCl .. I read some people needed to decross link for almost 24h when they fixed too long (the internet is full of ChIp-desperate students like us haha). About the 3kb fragment have you checked for contamination in your cells? Do you use salmon sperm DNA somewhere in your protocol for instance as a carrier? Or to avoid non specific binding during you chIP? Also ising the extraction kit helps gain time and procede to several tests. If I can give you an advice if you want to solve your issue : fresh start put one standard protocol (you can find a paper that focus on transcription factors and check their protocol) than start changing one parameter at a time! Also compare the different old protocols you used to gain time. Good Luck be patient and stubborn like your 3kb fragment!! ;)
AE is for Amplification efficiency ( that you have to evaluate for each primer couple) when you have residues of proteinase K or any kind of proteins, phenols etc you will have high AE which is a sign of a pcr inhibition. I had this issue because when you treat with proteinase K after the ON decrosslink this doesn't affect the gel migration (fragment analysis you have the good size etc) but it does affect the qpcr.. that is why in the diagenode protocole they keep the ON decrosslink for last so that you get rid of all the enzymes you used before. Hope this little change will fixe the issue :) Ghina
AE is for Amplification efficiency ( that you have to evaluate for each primer couple) when you have residues of proteinase K or any kind of proteins, phenols etc you will have high AE which is a sign of a pcr inhibition. I had this issue because when you treat with proteinase K after the ON decrosslink this doesn't affect the gel migration (fragment analysis you have the good size etc) but it does affect the qpcr.. that is why in the diagenode protocole they keep the ON decrosslink for last so that you get rid of all the enzymes you used before. Hope this little change will fixe the issue :) Ghina
May be proteinase treatment is the issue. Earlier, I used to do RNase and proteinase treatment before ON reverse crosslink and recently I changed it to ON crosslink followed by RNase and protease treatment (I might have seen this in some literature). What is AE?
May be proteinase treatment is the issue. Earlier, I used to do RNase and proteinase treatment before ON reverse crosslink and recently I changed it to ON crosslink followed by RNase and protease treatment (I might have seen this in some literature). What is AE?
Hi Ghina, I am happy that you got things working for you. Thanks for your inputs those are variations which I haven't tried so far. I will definitely give them a try and update with what happens. I had few questions: my goal is pull down transcription factors which are not directly bound to DNA and so I have to fix them for 15' though 10' give good sonication result but I can't see the desired enrichment through qPCR. Hence I use 15' but I tried 10' long time back and I have changed a lot ever since so probably I should give that another try. What are your target proteins? what do you mean by 7cycles? 30'On/30'off for 7' or 7 repetitions of 10' cycles? I fixed the issue with my DNA isolation, quality and gel, they are good now. My biggest problem is getting the right fragment size after sonication. I do get a nice fragments around 300-100 bp but unfortunately there is always a little bit of 3-2 kb fragment, which is just too stubborn to be broken down. After performing ChIP, even this 3-2 kb fragment gets enriched. May be the cross linking with NaCl might help I will give that also a try. Thanks again for your inputs
Hi Ghina, I am happy that you got things working for you. Thanks for your inputs those are variations which I haven't tried so far. I will definitely give them a try and update with what happens. I had few questions: my goal is pull down transcription factors which are not directly bound to DNA and so I have to fix them for 15' though 10' give good sonication result but I can't see the desired enrichment through qPCR. Hence I use 15' but I tried 10' long time back and I have changed a lot ever since so probably I should give that another try. What are your target proteins? what do you mean by 7cycles? 30'On/30'off for 7' or 7 repetitions of 10' cycles? I fixed the issue with my DNA isolation, quality and gel, they are good now. My biggest problem is getting the right fragment size after sonication. I do get a nice fragments around 300-100 bp but unfortunately there is always a little bit of 3-2 kb fragment, which is just too stubborn to be broken down. After performing ChIP, even this 3-2 kb fragment gets enriched. May be the cross linking with NaCl might help I will give that also a try. Thanks again for your inputs
Hi Ghina, I am still struggling with this problem. I tried using different concentrations of SDS in my sonication buffer and that seemed to help for the first time but unfortunately I am not successful in reproducing that result yet. It is pretty frustrating so far!!
Hi Ghina, I am still struggling with this problem. I tried using different concentrations of SDS in my sonication buffer and that seemed to help for the first time but unfortunately I am not successful in reproducing that result yet. It is pretty frustrating so far!!
well 115% is not perfect but ok I guess.. but if it's higher for your target genes you can play with the temperature (even though it has to do with the Tm of each couple you can use higher temperature like 60°C to reduce non specific binding if it doesn't work i think you have to change your primers) sometimes changing the primers quantity can work too .. I had like way more than 160% amplification when my qPCR was inhibited by the pk residues ... Once I left the ON decrosslink for last and thn amplified at 60°C instead of 55° C my efficiency dropped to 100-85% and I had only one amplification and a good curve.
well 115% is not perfect but ok I guess.. but if it's higher for your target genes you can play with the temperature (even though it has to do with the Tm of each couple you can use higher temperature like 60°C to reduce non specific binding if it doesn't work i think you have to change your primers) sometimes changing the primers quantity can work too .. I had like way more than 160% amplification when my qPCR was inhibited by the pk residues ... Once I left the ON decrosslink for last and thn amplified at 60°C instead of 55° C my efficiency dropped to 100-85% and I had only one amplification and a good curve.
Hi Saptha, I'm very happy this could help you... since our last discussion i found out I had to use the proteinase K before the ON decrosslink to get rid of it ....in fact residues of proteinase K inhibit your qPCR....may be this is what you have ? The ON decrosslink must be the last step of the "decrosslink process". I also use the extraction kit of activmotif with column to have clean samples.. any residue could inhibit your qpcr... hope this will help you I'm almost sure this is the issue... do you have like very very high AE? Ghina
Hi Saptha, I'm very happy this could help you... since our last discussion i found out I had to use the proteinase K before the ON decrosslink to get rid of it ....in fact residues of proteinase K inhibit your qPCR....may be this is what you have ? The ON decrosslink must be the last step of the "decrosslink process". I also use the extraction kit of activmotif with column to have clean samples.. any residue could inhibit your qpcr... hope this will help you I'm almost sure this is the issue... do you have like very very high AE? Ghina
Hi Ghina,
Thanks for the NaCl tip. That does wonder to the reverse crosslinking process. Now I am able to get the desired fragment length but now my ChIP-qPCR is not working. Would you mind sharing your entire ChIP protocol with me?
Hi Ghina,
Thanks for the NaCl tip. That does wonder to the reverse crosslinking process. Now I am able to get the desired fragment length but now my ChIP-qPCR is not working. Would you mind sharing your entire ChIP protocol with me?
More
VOTE
Hi Saptha,
I finally solved my issues it was the reversed cross-link, the DNA extraction quality and also the amount of EtBr in my gel. I now treat 1h with Rnase A at 37°C, reverse the cross-link ON 65°C with 0,2M NaCl (NaCl really changed the profile! ) then treat 1h with PK at 45°C, I am using ActivMotif kit to extract DNA for qPCR (with the Nanodrop I can see i have no phenol/Chlo contamination my DNA is clean) and now i can see my samples even with only 500ng on my gel also I now add more EtBr on my gel (10µL for like 150mL) . My fragments are about 200-400bp I use JLat cells (jurkat) I sonicate 10M cells (Diagenode kit medium SDS) , 7 cycles and I only fix 10'. Analysing the fragments size seems more complicated than the ChIp itself !! Hope this can help you out ! https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2077865/pdf/1746-4811-3-11.pdf I think more than 10 -15 cycles is way too much for jurkat cells probably like me ,you also have an issue after sonication : reverse cross link dna isolation , gel migration?! try to run sonicated and non sonicated samples on your gel +/- NaCl +/- PK and try a different DNA extraction technique 1µg-500ng should be ok try not to load too much on your gel ! Good luck!!
Hi Saptha,
I finally solved my issues it was the reversed cross-link, the DNA extraction quality and also the amount of EtBr in my gel. I now treat 1h with Rnase A at 37°C, reverse the cross-link ON 65°C with 0,2M NaCl (NaCl really changed the profile! ) then treat 1h with PK at 45°C, I am using ActivMotif kit to extract DNA for qPCR (with the Nanodrop I can see i have no phenol/Chlo contamination my DNA is clean) and now i can see my samples even with only 500ng on my gel also I now add more EtBr on my gel (10µL for like 150mL) . My fragments are about 200-400bp I use JLat cells (jurkat) I sonicate 10M cells (Diagenode kit medium SDS) , 7 cycles and I only fix 10'. Analysing the fragments size seems more complicated than the ChIp itself !! Hope this can help you out ! https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2077865/pdf/1746-4811-3-11.pdf I think more than 10 -15 cycles is way too much for jurkat cells probably like me ,you also have an issue after sonication : reverse cross link dna isolation , gel migration?! try to run sonicated and non sonicated samples on your gel +/- NaCl +/- PK and try a different DNA extraction technique 1µg-500ng should be ok try not to load too much on your gel ! Good luck!!
More
VOTE
Hi Saptha !
As I said in my first comment I am very new at that ChIp thing, all I know I get it from literature commercial protocols home made peotocols and through research gate questions haha!
My target proteins are histones that is why I can fix my cells only 10min from what I read when trying to solve my issues when you work on transcription factors or factors that do not directly bind on your dna you have to fix longer .. just as you do. I really don't know if this will impact sonication drasticallyI think you can find an answer for that in diagenode protocol at the very end of the booklet.
I sonnicate mine 7cycles 30sOn/30sOff I tried 9 don't see big difference and 10 is too much for me 100-300bp
NaCl really helps with reversing cross-link and if you fixed you cells 15min you will defently need NaCl .. I read some people needed to decross link for almost 24h when they fixed too long (the internet is full of ChIp-desperate students like us haha).
About the 3kb fragment have you checked for contamination in your cells? Do you use salmon sperm DNA somewhere in your protocol for instance as a carrier? Or to avoid non specific binding during you chIP?
Also ising the extraction kit helps gain time and procede to several tests. If I can give you an advice if you want to solve your issue : fresh start put one standard protocol (you can find a paper that focus on transcription factors and check their protocol) than start changing one parameter at a time! Also compare the different old protocols you used to gain time. Good Luck be patient and stubborn like your 3kb fragment!! ;)
Hi Saptha !
As I said in my first comment I am very new at that ChIp thing, all I know I get it from literature commercial protocols home made peotocols and through research gate questions haha!
My target proteins are histones that is why I can fix my cells only 10min from what I read when trying to solve my issues when you work on transcription factors or factors that do not directly bind on your dna you have to fix longer .. just as you do. I really don't know if this will impact sonication drasticallyI think you can find an answer for that in diagenode protocol at the very end of the booklet.
I sonnicate mine 7cycles 30sOn/30sOff I tried 9 don't see big difference and 10 is too much for me 100-300bp
NaCl really helps with reversing cross-link and if you fixed you cells 15min you will defently need NaCl .. I read some people needed to decross link for almost 24h when they fixed too long (the internet is full of ChIp-desperate students like us haha).
About the 3kb fragment have you checked for contamination in your cells? Do you use salmon sperm DNA somewhere in your protocol for instance as a carrier? Or to avoid non specific binding during you chIP?
Also ising the extraction kit helps gain time and procede to several tests. If I can give you an advice if you want to solve your issue : fresh start put one standard protocol (you can find a paper that focus on transcription factors and check their protocol) than start changing one parameter at a time! Also compare the different old protocols you used to gain time. Good Luck be patient and stubborn like your 3kb fragment!! ;)
More
VOTE
AE is for Amplification efficiency ( that you have to evaluate for each primer couple) when you have residues of proteinase K or any kind of proteins, phenols etc you will have high AE which is a sign of a pcr inhibition. I had this issue because when you treat with proteinase K after the ON decrosslink this doesn't affect the gel migration (fragment analysis you have the good size etc) but it does affect the qpcr.. that is why in the diagenode protocole they keep the ON decrosslink for last so that you get rid of all the enzymes you used before. Hope this little change will fixe the issue :)
Ghina
AE is for Amplification efficiency ( that you have to evaluate for each primer couple) when you have residues of proteinase K or any kind of proteins, phenols etc you will have high AE which is a sign of a pcr inhibition. I had this issue because when you treat with proteinase K after the ON decrosslink this doesn't affect the gel migration (fragment analysis you have the good size etc) but it does affect the qpcr.. that is why in the diagenode protocole they keep the ON decrosslink for last so that you get rid of all the enzymes you used before. Hope this little change will fixe the issue :)
Ghina
More
VOTE
Ok the efficiency of the qPCR reaction for test genes have a range of 115-160% whereas the house keeping gene falls in the range of 90-100%
Ok the efficiency of the qPCR reaction for test genes have a range of 115-160% whereas the house keeping gene falls in the range of 90-100%
More
VOTE
May be proteinase treatment is the issue. Earlier, I used to do RNase and proteinase treatment before ON reverse crosslink and recently I changed it to ON crosslink followed by RNase and protease treatment (I might have seen this in some literature).
What is AE?
May be proteinase treatment is the issue. Earlier, I used to do RNase and proteinase treatment before ON reverse crosslink and recently I changed it to ON crosslink followed by RNase and protease treatment (I might have seen this in some literature).
What is AE?
More
VOTE
Hi Ghina,
I am happy that you got things working for you. Thanks for your inputs those are variations which I haven't tried so far. I will definitely give them a try and update with what happens.
I had few questions: my goal is pull down transcription factors which are not directly bound to DNA and so I have to fix them for 15' though 10' give good sonication result but I can't see the desired enrichment through qPCR. Hence I use 15' but I tried 10' long time back and I have changed a lot ever since so probably I should give that another try. What are your target proteins?
what do you mean by 7cycles? 30'On/30'off for 7' or 7 repetitions of 10' cycles?
I fixed the issue with my DNA isolation, quality and gel, they are good now. My biggest problem is getting the right fragment size after sonication. I do get a nice fragments around 300-100 bp but unfortunately there is always a little bit of 3-2 kb fragment, which is just too stubborn to be broken down. After performing ChIP, even this 3-2 kb fragment gets enriched.
May be the cross linking with NaCl might help I will give that also a try.
Thanks again for your inputs
Hi Ghina,
I am happy that you got things working for you. Thanks for your inputs those are variations which I haven't tried so far. I will definitely give them a try and update with what happens.
I had few questions: my goal is pull down transcription factors which are not directly bound to DNA and so I have to fix them for 15' though 10' give good sonication result but I can't see the desired enrichment through qPCR. Hence I use 15' but I tried 10' long time back and I have changed a lot ever since so probably I should give that another try. What are your target proteins?
what do you mean by 7cycles? 30'On/30'off for 7' or 7 repetitions of 10' cycles?
I fixed the issue with my DNA isolation, quality and gel, they are good now. My biggest problem is getting the right fragment size after sonication. I do get a nice fragments around 300-100 bp but unfortunately there is always a little bit of 3-2 kb fragment, which is just too stubborn to be broken down. After performing ChIP, even this 3-2 kb fragment gets enriched.
May be the cross linking with NaCl might help I will give that also a try.
Thanks again for your inputs
More
VOTE
Hi Ghina,
I am still struggling with this problem. I tried using different concentrations of SDS in my sonication buffer and that seemed to help for the first time but unfortunately I am not successful in reproducing that result yet. It is pretty frustrating so far!!
Hi Ghina,
I am still struggling with this problem. I tried using different concentrations of SDS in my sonication buffer and that seemed to help for the first time but unfortunately I am not successful in reproducing that result yet. It is pretty frustrating so far!!
More
VOTE
well 115% is not perfect but ok I guess.. but if it's higher for your target genes you can play with the temperature (even though it has to do with the Tm of each couple you can use higher temperature like 60°C to reduce non specific binding if it doesn't work i think you have to change your primers) sometimes changing the primers quantity can work too .. I had like way more than 160% amplification when my qPCR was inhibited by the pk residues ... Once I left the ON decrosslink for last and thn amplified at 60°C instead of 55° C my efficiency dropped to 100-85% and I had only one amplification and a good curve.
well 115% is not perfect but ok I guess.. but if it's higher for your target genes you can play with the temperature (even though it has to do with the Tm of each couple you can use higher temperature like 60°C to reduce non specific binding if it doesn't work i think you have to change your primers) sometimes changing the primers quantity can work too .. I had like way more than 160% amplification when my qPCR was inhibited by the pk residues ... Once I left the ON decrosslink for last and thn amplified at 60°C instead of 55° C my efficiency dropped to 100-85% and I had only one amplification and a good curve.
More
VOTE
Hi Saptha,
I'm very happy this could help you... since our last discussion i found out I had to use the proteinase K before the ON decrosslink to get rid of it ....in fact residues of proteinase K inhibit your qPCR....may be this is what you have ? The ON decrosslink must be the last step of the "decrosslink process".
I also use the extraction kit of activmotif with column to have clean samples.. any residue could inhibit your qpcr... hope this will help you I'm almost sure this is the issue... do you have like very very high AE?
Ghina
Hi Saptha,
I'm very happy this could help you... since our last discussion i found out I had to use the proteinase K before the ON decrosslink to get rid of it ....in fact residues of proteinase K inhibit your qPCR....may be this is what you have ? The ON decrosslink must be the last step of the "decrosslink process".
I also use the extraction kit of activmotif with column to have clean samples.. any residue could inhibit your qpcr... hope this will help you I'm almost sure this is the issue... do you have like very very high AE?
Ghina
More
VOTE