Hi everyone,
I encountered an issue with histone modification ChIPseq library prep using the NEBNext Ultra DNA lib prep kit. Hope anyone here could help to troubleshoot this.
1. I sheared my DNA using a Covaris S220 sonicator, the fragment size after sonication was 300-400 bp.
2. Immunoprecipitation was done using unmodified H3 (Abcam, H3K4me3 (active motif), and H3K27me3 (active motif) antibodies.
3. I got a low yield of ChIP-enriched DNA from the H3K*me3, with a concentration range from 2-5 ng.
4. I then decided to use 2 ng of DNA for preparing the library. I optimized the PCR cycles prior to the library preparation (using the input DNA) and found that 11 cycles are suitable for 2 ng of starting material, i.e. library final conc. around 10-20 ng/uL, with average fragment size 600-700 bp.
When I did the lib prep using the ChIP-enriched DNA, I only got good results from the input and unmodified H3, with average fragment size at around 600 bp and concentration of 10-20 ng/uL. The H3K*me3 libraries concentration was very low, i.e. 0.08 - 0.4 ng/uL. I dilute my library to a final concentration of 30 uL.
I did the same experiment previously with a lower amount of starting material and using different library prep kit and got the exact same pattern: no problem with input and unmodified H3 samples and a lower concentration of H3K*me3 libraries.
Any ideas about why this could have happened, or how to troubleshoot it would be greatly appreciated.
PS: I use the ChIP technique to study the developmental process in a non-model plant. It is a bit of a challenge for me to check the enrichment with PCR due to minimal information regarding potential target loci/ gene-specific involves in the developmental process of this plant.
Cheers,
Dina
I encountered an issue with histone modification ChIPseq library prep using the NEBNext Ultra DNA lib prep kit. Hope anyone here could help to troubleshoot this.
1. I sheared my DNA using a Covaris S220 sonicator, the fragment size after sonication was 300-400 bp.
2. Immunoprecipitation was done using unmodified H3 (Abcam, H3K4me3 (active motif), and H3K27me3 (active motif) antibodies.
3. I got a low yield of ChIP-enriched DNA from the H3K*me3, with a concentration range from 2-5 ng.
4. I then decided to use 2 ng of DNA for preparing the library. I optimized the PCR cycles prior to the library preparation (using the input DNA) and found that 11 cycles are suitable for 2 ng of starting material, i.e. library final conc. around 10-20 ng/uL, with average fragment size 600-700 bp.
When I did the lib prep using the ChIP-enriched DNA, I only got good results from the input and unmodified H3, with average fragment size at around 600 bp and concentration of 10-20 ng/uL. The H3K*me3 libraries concentration was very low, i.e. 0.08 - 0.4 ng/uL. I dilute my library to a final concentration of 30 uL.
I did the same experiment previously with a lower amount of starting material and using different library prep kit and got the exact same pattern: no problem with input and unmodified H3 samples and a lower concentration of H3K*me3 libraries.
Any ideas about why this could have happened, or how to troubleshoot it would be greatly appreciated.
PS: I use the ChIP technique to study the developmental process in a non-model plant. It is a bit of a challenge for me to check the enrichment with PCR due to minimal information regarding potential target loci/ gene-specific involves in the developmental process of this plant.
Cheers,
Dina
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