GSM8741855: 473A ATAC ad 2.5; Homo sapiens; ATAC-seq
For CUT&RUN assay, 0.5x10^6  cells bound to Concanavalin A-coated beads were incubated with indicated primary antibodies and secondary antibody.  pA-MNase was used to digest chromatin/DNA bound by the antibodies-protein complex in CUT&RUN, followed by Proteinase K digestion and PCI extraction. For ATAC-seq, 50,000 cells were collected, then washed once by 1×PBS and tagmented in 1×TD buffer with 2.5 μl Tn5, 0.01% digitonin, and 0.3×PBS. The reactions were incubated at 37 °C for 30 min in a thermomixer with 300 rpm mixing. The tagmented DNA was purified using the DNA Clean & Concentrator-5 kit. CUT&RUN libraries were prepared from 15-20ng DNA using NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645S), according to the manufacturer's instructions. Postamplification libraries were size selected at 160-320bp (CUT&RUN) in length using Agencourt AMPure XP beads from Beckman Coulter and were quantified using the Library Quantification Kit from Illiumina (Kapa Biosystems, KK4603). All CUT&RUN Libraries were pooled to a final concentration of 10nM and sequenced single-end using the Illumina HiSeq 4000. ATAC-seq Libraries were amplified and adapter dimers and primer dimers were cleaned up.