GSM8242614: Rad21 CUT&RUN on purified Ngn2 transduced astrocytes at 2dpi- rep1; Mus musculus; OTHER
The YY1, FLAG, cohesin (Rad21) and H3K27Ac CUT&RUN assays were performed as previously described with specific modifications (PMID: 28079019). Briefly, 2-3.6 * 10^5 iNs were harvested, washed twice, and resuspended in wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete). Concavalin A beads (BioMag® Plus, Polyscience) were activated with bead activation buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Cells were incubated with 10 μl of activated beads for 10 minutes at room temperature. Following incubation, the beads were resuspended in a cold antibody buffer (2 mM EDTA in digitonin buffer) containing antibody (5 μg YY1 (D5D9Z) Rabbit mAb #46395, 2μg FLAG antibody (Sigma, #F3165-.2MG), 5μg Rad21 (Biozol, #GTX106012), 1 μg of H3k27Ac (Abcam #39133) and the mixture was incubated on a nutator overnight at 4°C. On the next day, the beads were washed twice and resuspended in 0.75 μl of pAG-Mnase in digitonin buffer (0.1% digitonin, ThermoFisher Scientific, in wash buffer), and incubated for 10 minutes at room temperature on a rotator. Later, beads were washed twice with cold digitonin buffer and then resuspended in 50 μl of digitonin buffer containing 1 μl of 100 mM CaCl2. The suspension was incubated for 2 hours at 4°C on a nutator. Following the incubation, 33 μl of STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A (Thermo Fisher Scientific), 50 μg/mL Glycogen) was added to each reaction, and the mixture was incubated for 30 minutes at 37°C. DNA extraction was performed using UltraPure Phenol:Chloroform:Isoamylalcohol (25:24:1, v/v, ThermoFisher Scientific) and precipitated with 100% ethanol, 1 μl of Glycogen, and 1/10th volume of 3M Sodium Acetate for 4-16 hours at -20°C. DNA was then dissolved in 10 μl of 1 mM Tris-HCl pH 8 and 0.1 mM EDTA. CUT&RUN libraries were prepared with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, E7645S) using 6-30 ng of fragmented DNA. The quality of the CUT&RUN libraries was evaluated using the Agilent 2100 Bioanalyzer system (Agilent). Library was sequenced using 2x50bp reads.