GSM6606961: E14 TKO cells, D4, biological replicate 2, H3K27me3 (CUT&RUN_EpiLC); Mus musculus; OTHER
The Cut&Run protocol was optimized from Skene and Henikoff, 2017. Concanavalin A beads (Polysciences) were activated in 1ml of Binding Buffer (20mM HEPES-KOH, 10mM KCl, 1mM CaCl2, 10mM MnCl2). Between 200 000 and 300 000 cells were aliquoted in 1ml of Wash Buffer (20mM HEPES-KOH, 150mM NaCl, 0.5mM Spermidine (Sigma) and 1X Complete EDTA-free (Roche) protease inhibitor cocktail) and 20µL of activated beads were added to the cells, followed by 5 min incubation at RT on a rotating wheel. Cells were collected on a magnetic rack; the supernatant was replaced by 400µL of primary antibody diluted to 1:200 in Wash Buffer complemented with 2mM EDTA and 0.02% Digitonin (Millipore). Cells were incubated for one hour at RT. Samples were then washed twice in Dig-Wash Buffer (Wash Buffer + 0.02% Digitonin) for 15 min at RT and incubated with 1:400 home-made pA-MNase in Dig-Wash for 15 min at RT. After two more washes, cells were resuspended in 150µl of Dig-Wash and cooled-down for five min at 0°C. For targeted digestion, CaCl2 was added to the cells to a final concentration of 2mM followed by 30 min incubation on ice. The reaction was stopped by adding 150µl of 2X STOP Buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.02% Digitonin, 100ng/ml RNase1, 250ng/ml glycogen) and incubating the samples at 37°C for 20 min before centrifugation at 4°C 16000g for 5 min. Samples were placed on a magnetic rack and supernatant was recovered in a low binding eppendorf tube. Following addition of 0.1% SDS and 0.17mg/ml Proteinase K, samples were incubated at 70°C for 30 min. Purified DNA was obtained by phenol/chloroform extraction and precipitated with 100% Ethanol incubation at -20°C for at least 20 min. The DNA pellet was washed in 85% ethanol, spun down and air-dried before being resuspended in 25µl of 1mM Tris-HCl pH8.0. Library preparation was made according to the manufacturer's instructions (Accel NGS 2S DNA library kit, Swift Biosciences). For library amplification, 12 PCR cycles were applied as followed: 98°C for 10 sec, 60°C for 15 sec and 68°C for one minute. Library size and quantification was assessed on Agilent 4200 Tapestation. C