Greetings everyone! I have very much enjoyed following this forum for the last few months. I am a graduate student who is making a foray into ChIP-seq. I have a question for the more experienced people out there whether they think that the ChIP-seq library I have generated is suitable for sequencing (Bioanalyzer trace below). We have an experienced next-gen sequencing facility in the institute where I work, however they have not worked up ChIP-seq before...
Briefly:
- ChIP with Histone PTM antibody
- Samples generated from 4x106 cells per IP, digestion to mainly mono-nucleosome with MNase prior to IP.
- ChIP verified by positive control regions in PCR prior to library generation
- Library generated using NEB-NEXT ChIP seq master mix kit for Illumina. NEB NExt adapters and multiplexed oligos used. (Adapter are loop).
- Samples are to be sequenced on the Hi-Seq using barcoded primers (aiming for 20x106 reads per sample).
I did a trial libray preparation with an Input sample and a ChIP sample. I was unable to quantify the DNA, so I used 37 uL of a 50uL elution of ChIP, keeping the remainder for PCR verification. I used 25uL of a 2% Input sample (which was too dilute for quantification on NanoDrop).
For the library preparation, size selection was performed post adapter ligation using a 2% agarose gel (with EtBr). I didn't clean up with the MinElute kit after adapter ligation to avoid carry through of adapter junk. All other purification steps were with Qiagen columns. I excised the region around 200bp from a gel pre PCR amplification. Realistically, I think I took about 175 to 220-230 bp.
I performed 18 cycles of PCR amplification (using universal primer and either index 2 or 4) followed by clean up with MinElute column and have attached the Bioanalyzer trace (DNA1000 ChIP). Included are some exome capture libraries, please ignore these, the libraries I generated for ChIP seq are numbers 11 and 12: Input and ChIP.
The trace is quite unusual to my understanding - the first small peak is at 89bp and probably represents primers (adapter should be around 120-130 from what I understand). Aside from the molarity of the libraries being quite low, they also have a curious double peak (190 and 230bp). We are uncertain what this represents!
There is also junk around the 400bp region - I think that this might be ssDNA from too many PCR cycles?
I have no idea if this is acceptable, or whether the library will sequence? The sequencing facility think that it is probably OK, and that if necessary the high and low 'junk' can be removed with a bead clean up, but they haven't come across anything quite the same as this library before. Any input would be greatly appreciated!!!!!!!
Cheers,
O
Briefly:
- ChIP with Histone PTM antibody
- Samples generated from 4x106 cells per IP, digestion to mainly mono-nucleosome with MNase prior to IP.
- ChIP verified by positive control regions in PCR prior to library generation
- Library generated using NEB-NEXT ChIP seq master mix kit for Illumina. NEB NExt adapters and multiplexed oligos used. (Adapter are loop).
- Samples are to be sequenced on the Hi-Seq using barcoded primers (aiming for 20x106 reads per sample).
I did a trial libray preparation with an Input sample and a ChIP sample. I was unable to quantify the DNA, so I used 37 uL of a 50uL elution of ChIP, keeping the remainder for PCR verification. I used 25uL of a 2% Input sample (which was too dilute for quantification on NanoDrop).
For the library preparation, size selection was performed post adapter ligation using a 2% agarose gel (with EtBr). I didn't clean up with the MinElute kit after adapter ligation to avoid carry through of adapter junk. All other purification steps were with Qiagen columns. I excised the region around 200bp from a gel pre PCR amplification. Realistically, I think I took about 175 to 220-230 bp.
I performed 18 cycles of PCR amplification (using universal primer and either index 2 or 4) followed by clean up with MinElute column and have attached the Bioanalyzer trace (DNA1000 ChIP). Included are some exome capture libraries, please ignore these, the libraries I generated for ChIP seq are numbers 11 and 12: Input and ChIP.
The trace is quite unusual to my understanding - the first small peak is at 89bp and probably represents primers (adapter should be around 120-130 from what I understand). Aside from the molarity of the libraries being quite low, they also have a curious double peak (190 and 230bp). We are uncertain what this represents!
There is also junk around the 400bp region - I think that this might be ssDNA from too many PCR cycles?
I have no idea if this is acceptable, or whether the library will sequence? The sequencing facility think that it is probably OK, and that if necessary the high and low 'junk' can be removed with a bead clean up, but they haven't come across anything quite the same as this library before. Any input would be greatly appreciated!!!!!!!
Cheers,
O
