GSM6625561: Retina, Fbxl11-CKO1, P1, H3K36me2; Mus musculus; OTHER
Control or Fbxl11-CKO retinal cells at P1 (1 x 105 cells/sample) were mixed with human colorectal cancer cell line SW480 (1 x 104 cells/sample), and resuspended in buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine (Nacalai Tesque), 1x Protease Inhibitor Cocktail) at 1 x 105 cells/100 μl. Concanavalin A magnetic beads (Bangs Laboratories, USA) were mixed with the cells in binding buffer  (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) and rotated for 20 minutes at room temperature. Then, the cells were resuspended in 100 μl of antibody buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine (Nacalai Tesque), 1x Protease Inhibitor Cocktail, 0.05% Digitonin (Millipore), 2 mM EDTA, and 0.1% bovine serum albumin) containing antibodies listed below at 10 μg/mL and rotated overnight at 4℃. The cells were washed and resuspended in 100 μl Dig-wash Buffer containing 10 μg/ml anti-rabbit IgG antibody (Rockland Immunochemicals, USA, 611-201-122), rotated for 1 hr at room temperature, and resuspended in Dig-300 Buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 1x Protease Inhibitor Cocktail) containing 0.8 μl pAG-Tn5 adapter complex and rotated for 1 hr at room temperature. The cells were washed with Dig-300 Buffer three times, resuspended in 100 μl Tagmentation Buffer (Dig-300 Buffer containing 10 mM MgCl2), and incubated for 1 hr at 37℃. Then 2 μl of 10 mg/mL Proteinase K was added and further incubated at 37℃ for 30 min. DNA was purified by Phenol-Chloroform protocol and dissolved in 25 μl water. The DNA was subjected to PCR (72℃ (5 mins) x 1, 98℃ (30 sec) x 1, 98℃ (10 sec)/ 63℃ (15 sec) x 17, 72℃ (1 min) x 1) using i5 and i7 primers and Q5 High-Fidelity 2x Master Mix (New England Biolabs, USA). The library was sequenced with Novaseq6000.