GSM9084181: shRen_IgG_3; Mus musculus; OTHER
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. Following EpCAM enrichment, tumor cells were counted and resuspended at a concentration of 2 × 106 cells in wash buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM spermidine; protease inhibitor cocktail). For each CUT&RUN reaction, 2 × 105 cells were used. Cells were incubated with 10 µL of activated Concanavalin A-coated magnetic beads (Polysciences) for 10 minutes at room temperature on a hula mixer with constant agitation. Following bead binding, samples were placed on a magnetic stand to remove the supernatant. Bead-bound cells were resuspended in 100 µL of antibody buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 0.01% [w/v] digitonin; 2 mM EDTA; protease inhibitor), and primary antibodies were added as follows: anti-YAP1 ( Cell Signaling Technology, #14074), anti-TEAD1 (Cell Signaling Technology, #12292), anti-H3K27Ac (Abcam, ab4729), and anti-H3K4me1 (Cell Signaling Technology, #5326). A species-matched IgG was used as a negative control. Samples were incubated overnight at 4°C on a hula mixer set at a 50° tilt and 1 RPM. The next day, samples were washed twice with digitonin wash buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 0.01% digitonin; protease inhibitor). Protein A/G-MNase (pAG-MNase; 1 µg/mL) was added in digitonin wash buffer, and samples were incubated for 1 hour at 4°C with gentle agitation. After incubation, cells were washed three times with digitonin wash buffer and twice with low-salt buffer (20 mM HEPES, pH 7.5; 0.5 mM spermidine; 0.01% digitonin). Nuclease activation was performed by transferring the samples to calcium-containing buffer (3.5 mM HEPES, pH 7.5; 10 mM CaCl₂; 0.01% digitonin; protease inhibitor) and incubating on ice for 1 hour with continuous mixing. Cleaved DNA fragments were released by incubation at 37°C for 30 minutes in STOP buffer (170 mM NaCl; 20 mM EGTA; 0.01% digitonin; 50 µg/mL RNase A; 25 µg/mL Glycogen Blue). Supernatants were collected, and DNA was purified by phenol-chloroform extraction. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, #E7645) following the manufacturer's protocol. Library quality was assessed using the Fragment Analyzer (Agilent Technologies), and equimolar pooling of libraries was performed. Pooled libraries were gel purified, quantified on an Agilent 2100 Bioanalyzer, and sequenced as 75 bp single-end reads on an Illumina NextSeq 500 platform.