GSM8062062: LNCaP-shNT-2; Homo sapiens; ATAC-seq
Nuclei were isolated with nuclear lysis buffer (10 mM tris, 10 mM NaCl, 3 mM MgCl2, and 0.5% IGEPAL-630) and centrifuged at low speeds. The nuclei were resuspended in 50uL of transposase reaction mixture (22.5uL nuclease-free water, 25uL of TD buffer and 2.5uL of TDE1 enzyme, Illumina #20034197). Transposition was carried out at 37°C for 30 min, followed by DNA purification with DNA Clean and Concentrator-5 kit (Zymo Research) according to the manufacturer's recommendation An input of 50,000 cells was first collected by low speed centrifugation (500xg), followed by washing with 1x PBS. To lyse the cells, 50 ul lysis buffer was used to suspend the cells. The cells were then incubated on ice for 5 min after brief vortexing, followed by centrifugation at 500xg at 4°C. Immediately after removal of the lysis buffer supernatant, the obtained nuclei pellet was re-suspended in the transposition reaction mix containing the Tn5 transposase, and incubated for 30 min at 37°C. After the incubation step, the reaction mix was immediately purified with the Zymo DNA Clean & Concentrator kit to stop and remove any residual Tn5 activity. A subsequent PCR amplification step with limited number of cycles was used to amplify the DNA and finish off library construction