Hi everyone,
I'm supposed to do a lot of ChIP-Seq experiments in the coming weeks, I've optimized my first set in terms of qPCR, so my ChIP should be working. I'll also be doing the library prep using the NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) along with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645L). The samples will be sequenced on Novaseq 6000 S4, PE150, 30 million reads / sample.
I have the following conditions:
Untreated - IP, IgG, INPUT
Treated - IP, IgG, INPUT
I've got a couple of questions now regarding the library prep:
1. Is there a maximum number of libraries that I can pool together? In other words, are there any restrictions or things that I need to consider before pooling them?
What I know so far is that they need to have unique indices and since I'm using NEBNext Mulitplex Oligos for Illumina Set1, there are 12 different indices so I was considering having every 6 libraries pooled together (1 biological repeat).
2. Is it better to have the 2 biological replicates' libraries pooled together or to have each replicate's set separately?
3. Do I need to use the same quantity of DNA for IP, IgG and INPUT when I start the library preparation step? I will quantify them by Qubit before that. I usually get around 1-1.5 ng of DNA for the IP & IgG samples and approx. 100-200 ng DNA for the input. If I use the same quantity of DNA, would I still sequence the input for the same depth as the IgG & IP samples?
Thank you in advance!
I'm supposed to do a lot of ChIP-Seq experiments in the coming weeks, I've optimized my first set in terms of qPCR, so my ChIP should be working. I'll also be doing the library prep using the NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) along with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645L). The samples will be sequenced on Novaseq 6000 S4, PE150, 30 million reads / sample.
I have the following conditions:
Untreated - IP, IgG, INPUT
Treated - IP, IgG, INPUT
I've got a couple of questions now regarding the library prep:
1. Is there a maximum number of libraries that I can pool together? In other words, are there any restrictions or things that I need to consider before pooling them?
What I know so far is that they need to have unique indices and since I'm using NEBNext Mulitplex Oligos for Illumina Set1, there are 12 different indices so I was considering having every 6 libraries pooled together (1 biological repeat).
2. Is it better to have the 2 biological replicates' libraries pooled together or to have each replicate's set separately?
3. Do I need to use the same quantity of DNA for IP, IgG and INPUT when I start the library preparation step? I will quantify them by Qubit before that. I usually get around 1-1.5 ng of DNA for the IP & IgG samples and approx. 100-200 ng DNA for the input. If I use the same quantity of DNA, would I still sequence the input for the same depth as the IgG & IP samples?
Thank you in advance!
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