In regular ChIP-seq library preparation, my lab use 10ng as starting materials. Now I am studying a novel TF which just binds to several promters. This TF only transactivates 3-5 genes based on the preliminary data. 10ng DNA means I need to start with 10^10cells, which is impossible in my lab. Can I just start with 10^8 cells as regular ChIP-seq and do the library preparation?
In the regular ChIP-seq, although 10ng DNA is a lot, to a specific gene, the amoount of DNA is still very low. In my case, the amount of DNA for library preparation is low, but to a specific gene, it may be enough.
I know this question sounds silly and weird...
Thank you guys.
Exactly I agree with David. You can definitely prepare a library using either Biooscientific kits or illumina's . But you will encur alot of PCR duplications/artifacts due to lack of libarary complexity. Remember ChIP-Seq is traditionally used to assess the effects of the transcriptional regulation at the the genome wide level. You would essentially be wasting your time and resources if you are interested in a few genes.
You can worth try it. At least I have done T.F. ChIP seq and started library prep with 1ng or 100pg DNA using Illumina Highseq protocol. My library look great. and sequencing data look great as well. I hope you will get some motivation to do ur library prep.
Wish you good luck
Your TF of interest transactivates 3-5 genes, makes hard to understand. I headed human ENCODE for past 7 yrs and never encountered a single TF that has such a low number of target sites. I am sure you have lots more to find as you get a good Seq result. Surely more than 3/5.
Also we typically get 8-10 ng of DNA from 50M cells(suspension cells) and that should be enough to make a lib . Use Nextera kit from EPICENTER(now Illumina) Its a Tagmentation based chemistry and even though says need 50 ng to start, we worked perfectly with 5-6 ng and got good Seq Library.
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