GSM9084204: [ATAC-Seq] shRen_3; Mus musculus; ATAC-seq
Tumor-bearing livers were minced and digested in 15 mL of 1× PBS containing 7.5 mg Pronase, 7.5 mg Collagenase P, and 1.5 mg DNase I using a C Tube (Miltenyi Biotec) and the gentleMACS Octo Dissociator (Miltenyi) for 45 minutes at 100 rpm and 37 °C. Following digestion, the cell suspension was filtered through a 100 µm cell strainer and washed with an appropriate volume of Advanced DMEM/F12 (AdDMEM/F12) medium. Red blood cell lysis was performed using the Red Blood Cell Lysis Solution (Miltenyi, #130-094-183), followed by debris removal (Miltenyi, #130-109-398) and dead cell removal (Miltenyi, #130-090-101) according to the manufacturer's protocols. Cells were then incubated with Fc receptor blocking reagent (Miltenyi) for 10 minutes on ice. Tumor cells were labeled with anti-EpCAM-APC antibody (eBioscience, #17-5791-82; 1:100) for 30 minutes on ice. After washing, cells were incubated with anti-APC microbeads (Miltenyi) for an additional 30 minutes. Labeled cells were then loaded onto LS columns (Miltenyi) and processed according to the manufacturer's instructions. Positively labeled EpCAM⁺ cells were eluted and further processed for downstream applications. A total of 5 × 10^4 cells were pelleted in a pre-wetted 1.5 mL low-binding tube by centrifugation at 500 × g for 5 minutes at 4°C using a fixed-angle rotor. Cells were resuspended in 50 µL ice-cold ATAC-Resuspension Buffer (RSB: 10 mM Tris-HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl₂) supplemented with 0.1% (v/v) NP-40, 0.1% (v/v) Tween-20, and 0.01% (w/v) digitonin. The suspension was pipetted up and down three times and incubated on ice for 3 minutes to lyse the cells. To remove the lysis reagents, 1 mL ice-cold RSB containing 0.1% Tween-20 (without NP-40 or digitonin) was added, and the tube was gently inverted three times. Nuclei were pelleted by centrifugation at 500 × g for 10 minutes at 4°C, and the supernatant was carefully removed. Nuclei were resuspended in 50 µL of transposition reaction mix containing: 25 µL 2× TD buffer (Illumina), 2.5 µL Tn5 transposase (to reach 100 nM final concentration), 16.5 µL 1× PBS, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, 5 µL nuclease-free water. The mix was pipetted up and down six times to ensure homogeneous suspension. Samples were incubated at 37°C for 30 minutes at 1000 rpm using a thermomixer. Following transposition, DNA was purified using the Zymo DNA Clean & Concentrator-5 Kit (Zymo Research) according to the manufacturer's instructions and amplified by PCR.
GEO
GSM9084204_r1