ChIPSeq results NEB kit and MiSeq run settings

[BioGenomics]

Hi all,

Trying yet another protocol on our new MiSeq... We choose to go ahead with the NEB ChIPSeq lib prep kit (Ultra DNA in fact), but had some mixed library prep results:

--> After size selection using SeraMag beads (not AMPure), we have only a very low band around 150 bp, most likely adaptors dimers/concatemers (low input ChIPed DNA). However, the supernatant seems to have the size we need (200-350 bp). See pictures attached in pdf. Should we go for gel selection or just use the supernatant as input for sequencing ? A WGA test does yield the correct size after cleanup though (right side)

--> I assume the safest way to really quantify this is to go for the KAPA qPCR library quantification right ?

--> At which settings should the MiSeq be run with the NEB kit ? Sample sheet according to TrueSeq LT right ? SE run WITH indexing correct ? I had trouble finding all info on the Indexing kit sold by NEB... Which concentration for ChIP libs sequencing ? I was planning 12pM with 5 % PhiX

thanks for any insights here !

Cheers

[BioGenomics]

Hi all,

Still looking for feedback whether or not we should go on with the faint smear seen on the gels (we started with 0.25 ng/ul bacterial ChipEd DNA)

thanks