Hello there,
I want to do ChIP-seq for H3K4me modified histone and have a native chromatin extraction protocol with micrococcal nuclease step in place. I plan to use the Illumina HiSeq200 for Paired end sequencing of 58bp-long reads.
I understand that the best fragment size range for this approach is 200 – 300 bp.
What I don’t understand is how a micrococcal nuclease digestion step can be good for ChIP-seq since the resulting fragment size range is quite broad (mono- di and tri- nucleosomal band being abundant) and the sample library prep prior to sequencing requires a very narrow size range.
Moreover, does anyone know whether sonication instead of micrococcal digestion is ok when the subject of study is the histone or its modifications?
Finally, as I want to study T cells from blood, I can obtain around 20 million T cells and am not sure whether this will be enough for sequencing after ChIP.
Can anyone help? Any protocol in place?
Thanks in advance
Alex
I want to do ChIP-seq for H3K4me modified histone and have a native chromatin extraction protocol with micrococcal nuclease step in place. I plan to use the Illumina HiSeq200 for Paired end sequencing of 58bp-long reads.
I understand that the best fragment size range for this approach is 200 – 300 bp.
What I don’t understand is how a micrococcal nuclease digestion step can be good for ChIP-seq since the resulting fragment size range is quite broad (mono- di and tri- nucleosomal band being abundant) and the sample library prep prior to sequencing requires a very narrow size range.
Moreover, does anyone know whether sonication instead of micrococcal digestion is ok when the subject of study is the histone or its modifications?
Finally, as I want to study T cells from blood, I can obtain around 20 million T cells and am not sure whether this will be enough for sequencing after ChIP.
Can anyone help? Any protocol in place?
Thanks in advance
Alex
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