Hi,

We are using the following standard ATAC protocol with minor changes: Article ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide

Though ATAC seq efficiency vary across cell types because of different chromatin organisation. Such, cells with easily accessible big regions (like stem cells) tend to give good results (slight waves on the Bioanalyzer and sharp peaks in the promoters after sequencing); but such cells like cardiomyocytes (which have tight chromatin and nuclei, embedded in EM), tend to give bad results.

Finally, wavy structure of the library (basically due to nucleosome localisation), is not necessary a sign of a good ATAC library, and vice versa. We received only background in super-wavy libraries and nice peaks in the libraries which had no waves at all.

Best,

Michail

Hi Shubhang,

in addition to the new protocol, did you test your primers with in silica PCR?

if not, and it depends on your sample species, you can use Primer-Blast from NCBI or in-silico-PCR from the UCSC. if it works on those sites, no way it won't work in your study?

fred

Hi Ann Delaforest

The traces do not look perfect, but may work out. I would suggest to sequence 1-2 samples and look at the reads distribution. If you will see good peaks, go ahead and sequence the rest.

Good luck,

Michail

Hi Michail Yekelchyk Shubhang Bhalla , Sorry to bother you here. Actually I asked it other place but didn't get answer yet. Please help me if you can.

I am a bit confused about the ATAC-seq primer design as most of the place it isn't explained in detail. Mostly they mentioned Custom Nextera PCR Primer 1 and 2 (Contains Barcode). But I am not getting any clue how to design it.

Hi Md Saimoon Rahman ,

We usually use the primers from Illumina Nextera kit, which have known sequences (attached). So there is no need to re-invent the bicycle and design them from scratch. If you want to order the primers by your own, you can use the sequences from here (attached), for example.

Good luck!

Michail