GSM8615532: H3K27me3-wt3; Mus musculus; OTHER
Murine lung EC were subjected to FACS, pelleted for 5 min at 300 × g at 4°C and resuspended in 50 µl of cold complex formation buffer (1xCB) (50 mM Tris-HCL, pH 7.5; 150 mM NaCl; 0.05% Triton X-100; 12.5% glycerol). The cells were permeabilized for 10 min on ice. For each antibody, 0.16 µg of transposase was incubated with 3.3 µM adapter sequence in 1x CB buffer for 10 min. After adapter loading, 1 µg of antibody (H3K27ac, ab4729; H3K4me1, ab8895; H3K4me3, ab8580; H3K27me3, Cell Signaling, C36B11; H3K9me3, abab8898) was added, and the mixture was incubated for 30 min at room temperature. The transposon-adapter-antibody complex was added to the permeabilized cell suspension and incubated for 1 h at room temperature. In parallel, 7.5 x 102 permeabilized yeast nuclei were incubated with H2B (Hölzel Diagnostika, BOS-M30930) targeting the transposase complex. After 1 h of incubation, the yeast-transposase-H2B antibody mixture was added to the murine cell suspension, which was subsequently washed 3 times with washing buffer (50 mM Tris-HCL (pH 7.5), 150 mM NaCl, and 0.05% Triton X-100). The cell pellet was resuspended in 50 µl of washing buffer, and 1 µl of 1 M MgCl2 was added to initiate the transposition reaction. The solution was incubated for 1 h at 37°C. The transposase reaction was stopped by adding 4 µl of 0.5 M EDTA (pH 8), 2 µl of 10% SDS, and 1 µl of proteinase K (20 mg/ml; Qiagen), and the mixture was incubated for 1 h at 55°C following DNA purification with a MinElute PCR Purification Kit (Qiagen). The DNA was eluted 2x with 12 µl of elution buffer. The DNA eluate was PCR amplified using 1x NEBNext High Fidelity Mix in a total volume of 50 µl. ACT libraries were purified with a 1.4x volume of Ampure XP DNA purification beads, and the DNA was eluted in 12 µl of elution buffer.