GSM8778738: WT_ES_H3K4me2_D5-BMP4_Rep2; Mus musculus; OTHER
We performed CUT&Tag according with the protocol described in (Kaya-Okur et al., 2020), closely following the procedure described in “Bench top CUT&Tag V.2” (S Kaya-Okur and Henikoff, 2019). We used 500 000 per sample cells per sample and the anti-FLAG (Sigma-Aldrich F3165-5MG; 1/50) for CUT&Tag on transcription factors, or 200 000 nuclei that were previously fixed and frozen following the procedure described in(Kaya-Okur et al., 2020) with anti-H3K4me2 (Abcam ab176878; 1/100), anti-H3K27me3 (Cell Signaling 9733S; 1/100) primary antibodies. The primaries antibodies were incubated overnight at 4ºC on a rotator. Samples were then incubated with a secondary anti-rabbit IgG (ABIN6923140) or anti-mouse IgG (ABIN6923141) at room temperature on a rotator. After an incubation of 1h at room temperature with a homemade purified pA-TN5 complex, the Tn5 enzyme tagmentation reaction was performed by adding a buffer containing MgCl2 to the samples and incubating for 1h at 37°C. To stop tagmentation and solubilize DNA fragments, we added 10 μL 0.5M EDTA, 3 μL 10% SDS and 2.5 μL 20 mg/mL Proteinase K to each sample and samples were incubated at 50°C for 1h. Purified DNA was obtained by phenol/chloroform extraction and precipitated with 100% ethanol and centrifugation. The DNA pellet was washed in 80% ethanol, spun down and air-dried before being resuspended in 25µl of 1mM Tris-HCl pH8.0. Nextera primers or indexed primers were used to perform PCR. Library quality was assessed using 4200 TapeStation system (Agilent) and paired-end, 100 bp sequencing on a DBNSEQ-G400 was performed by BGI. All information is included as columns in the SAMPLES section above All information is included as columns in the SAMPLES section above