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No DNA in ChIP IgG Control - Strange?
Hi Guys,
Thanks in advance for your help!
Background:
It's my first time doing ChIP. I am testing a native ChIP protocol with MNase digestion using Protein A/G beads and H3K4Me3 and H3K27Me3. Because my samples are mononucleosomes (140bp) I didn't do qPCR prior to library preparation because of the difficulty of designing primers for such small fragments. I used 30,000 Cells (10,000 per IP).
Problem:
Although there is product in my H3K4Me3 and H3K27Me3 IP's, I have no product in my IgG sample (as detectable by HS Bioanalyzer). In my naive knowledge of ChIp I am tempted to say this is a great result as my IgG is a negative control and should hypothetically have pulled down no sample but someone else in my lab who has done X-linked ChIP using Diagenode's kit has a fair deal of product in her H3K's AND IgG's.
Any ideas as to the difference? I used Rabbit IgG instead of mouse (my cells are murine) - would that make a difference?
I've attached my Bioanalyzer traces.
Side note: My Input control is quite low - the main kit I am modifying for this N-ChIP suggests using 10ul from a 100ul dilution of chromatin for Input control. Instead, I pooled 4ul of each Input dilution prior to immunoprecipitation to result in 12ul of Input control for library prep. Do you think this is a good method for Input control? I am quite worried that the concentration is lower than my IPs - although it makes sense why it is (due to the lower input) I am not sure this is a good experimental design or if it is normal for all ChIPs? Would a better design be splitting my chromatin in 4 - 3 x 100ul for IPs and 1x 100ul for Input control? That way the input is the same - or does that not matter since I only pull down a percentage of DNA in each 100ul IP vs ''100%'' of on input control?
Sorry for the rambling questions!
Thanks in advance for your help!
Background:
It's my first time doing ChIP. I am testing a native ChIP protocol with MNase digestion using Protein A/G beads and H3K4Me3 and H3K27Me3. Because my samples are mononucleosomes (140bp) I didn't do qPCR prior to library preparation because of the difficulty of designing primers for such small fragments. I used 30,000 Cells (10,000 per IP).
Problem:
Although there is product in my H3K4Me3 and H3K27Me3 IP's, I have no product in my IgG sample (as detectable by HS Bioanalyzer). In my naive knowledge of ChIp I am tempted to say this is a great result as my IgG is a negative control and should hypothetically have pulled down no sample but someone else in my lab who has done X-linked ChIP using Diagenode's kit has a fair deal of product in her H3K's AND IgG's.
Any ideas as to the difference? I used Rabbit IgG instead of mouse (my cells are murine) - would that make a difference?
I've attached my Bioanalyzer traces.
Side note: My Input control is quite low - the main kit I am modifying for this N-ChIP suggests using 10ul from a 100ul dilution of chromatin for Input control. Instead, I pooled 4ul of each Input dilution prior to immunoprecipitation to result in 12ul of Input control for library prep. Do you think this is a good method for Input control? I am quite worried that the concentration is lower than my IPs - although it makes sense why it is (due to the lower input) I am not sure this is a good experimental design or if it is normal for all ChIPs? Would a better design be splitting my chromatin in 4 - 3 x 100ul for IPs and 1x 100ul for Input control? That way the input is the same - or does that not matter since I only pull down a percentage of DNA in each 100ul IP vs ''100%'' of on input control?
Sorry for the rambling questions!