title: GSM8282485: MITF-WT-Flag; Homo sapiens; OTHER
library_name: GSM8282485
library_construction_protocol: 5.105 cells were then harvested using scrapers, centrifuged at 600 g for 5 minutes, and washed twice with washing buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and 1X protease inhibitor cocktail). Afterward, cells were immobilized on pre-activated Concanavalin A-coated magnetic beads (EpiCypher #Cat 21-1401) at 4°C for 10 minutes. Incubation with anti-Flag (Sigma, #F3165) or Rabbit IgG (Cell Signaling, #3900S) in antibody buffer (washing buffer supplemented with 2 mM EDTA and 0.025% digitonin) followed, held overnight at 4°C with rotation. The following day, cells were washed with a washing buffer containing 0.025% digitonin. Subsequently, pAG-MNase enzyme (500 μg/mL) was introduced, and CaCl2 at 2mM final concentration was added to activate the enzyme at 0°C for 30 minutes. The reaction was terminated using 2X Stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen, and 500 pg/mL E. coli spike-in control). The Protein-DNA complex was released by undergoing incubation with the Proteinase K (1 μL/mL, Thermo Fisher Scientific, #EO0491) for 1 hour at 50°C. DNA fragment purification utilized the Genomic DNA Clean & Concentrator kit (ZymoResearch, #D465). CUT&RUN experiments were conducted in triplicate. Illumina's NEBNext Ultra II library preparation reagents (E7645L) Cut and Run