ATAC-qPCR

ATAC_qPCR

1. Dissect embryos in Ringers solution. Single neural folds (˜3000 cells) contains enough cells for testing a few primer pairs.
2. Transfer dissected samples to a microcentifuge tube (low binding).
3. Rinse with 300uL Accumax to remove excess salt from Ringers
4. Add 300uL of Accumax and incubate the tissue at RT for 20-30’, rocking at orbital shaker. During this period, gently pipette the tissue up and down to help tissue start dissociate.
5. Centrifuge 3 min 600rcf at room temperature and withdraw liquid.
6. During dissociation, prepare necessary buffers.
7. Wash the cells once with ATAC-RSB. Centrifuge 3 min 600rcf at room temperature.
8. Remove supernatant.
9. Resuspend with 50 µl ATAC-RSB-LYSIS, incubate on ice for 3 minutes.
10. Wash by adding 1 mL ATAC-RSB-WASH, mix by inversion.
11. Centrifuge for 5 minutes at 500rcf 4℃.
12. Remove supernatant.
13. Resuspend in 50µl of OMNI-ATAC Mix.
14. Incubate for one hour at 37℃ on heatblock, mix at 500rpm.
15. Cleanup with pre-amp Qiagen Minielute DNA columns (21µl 55℃ elution).

16. Tagmented DNA can be used for a standard qPCR protocol. Ex: 

    Primers* (fwd + rev) 100uM - 1ul

    DNA - 2ul
    SyBR green - 5ul 

    H2O - 2ul

17. For the analysis, run a ΔΔCT, where raw CT values from target regions are first normalized to a control region defined by the absence of transcription factor binding, no enrichment of active histone marks and low DNA accessibility in available CUT&RUN and ATAC datasets. Changes in chromatin accessibility in treated samples are subsequently compared to control samples.

* Primers should be designed following standard qPCR primers guidelines: lengths of 18–30 bases, a GC content of 50–60%, and a melting temperature between 60-63oC and the amplicon size should be 70–150 bp for maximum efficiency, and primers must be designed to avoid secondary structures, self-dimers, and 3' complementarity. Useful tool : https://www.primer3plus.com/index.html