title: GSM5006033: 2 CUT&RUN Sox2; Mus musculus; OTHER
library_construction_protocol: Cut&Run was performed as previously described (Skene et al. 2018) with some modifications. The number of samples was determined according to the number of antibodies to be tested and the number of replicates (2 replicates per antibody and 2 replicates for IgG). The appropriate volume of Concanavalin A magnetic beads (10 μl per sample) were mixed into 1.5 ml of ice-cold Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1mM MnCl2) and placed on a magnetic stand for 2 min. The supernatant was removed, and the beads were washed with 1.5 ml Binding Buffer. After magnetic separation of the beads, the supernatant was removed, and a volume of Binding Buffer equal to the initial beads volume (10 μl per sample) was added to the beads. The beads were placed on ice.    E13.5 ganglionic eminences were isolated from embryos in L15 medium (6 Embryos per genotype). Tissues were pooled for each genotype, triturated by pipetting, and filtered through a cell-strainer capped 5 ml polystyrene round- bottom tube (BD Falcon) to generate single-cell suspensions. Cells were counted using the Luna Automated Cell Counter (Logos Biosystems). The appropriate volume of cells to obtain 250,000 cells per sample was centrifuged at 600 g for 3 min at 4°C. The supernatant was removed and the cell pellet was gently resuspended in ice-cold Wash Buffer (20mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail). The cells were centrifuged at 600 g for 3 min at 4°C. The supernatant was removed and cells were gently resuspended in 1 ml of Wash Buffer. The Concanavalin A bead suspension was added to the cells, while gently vortexing (~1000 rpm), and the tube was incubated with rotation for 10 min at 4°C. The cells/beads suspension was split into aliquots, according to the number of samples previously determined. Tubes were placed on a magnetic stand and the supernatant removed. 50 ul of Antibody Buffer (20mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 2 mM EDTA, 0.02% digitonin, EDTA-free protease inhibitor cocktail) containing 0.5 μg of antibody was added to each tube, while gently vortexing. The samples were incubated with rotation for 2 hours at 4°C. The samples were centrifuged for 5 seconds at 1000 rpm, and placed on the magnetic stand. The supernatant was removed, and the pellet resuspended gently in 1 ml ice-cold Digitonin Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.02% digitonin, EDTA-free protease inhibitor cocktail). The Digitonin Buffer wash was repeated once. Tubes were placed on the magnetic stand, the supernatant removed and 50 μl of pA-Mnase solution (final concentration 700 ng/ml in Digitonin Buffer) were added to each tube while gently vortexing. The samples were incubated with rotation for 1 hours at 4°C. The samples were centrifuged for 5 sec at 1000 rpm and placed on the magnetic stand. The supernatant was removed and the pellets were washed in ice-cold Digitonin Buffer two times, as described above. After removal of supernatant from washes, 150 μl of Digitonin Buffer were added to each sample while gently vortexing. Tubes were placed on a metal block on ice (0°C) for 5 min. Tubes were removed from ice briefly to add 3 μl 100 mM CaCl2 while gently vortexing, then tubes were returned to 0°C for 30 min. 100 ul of Stop Buffer (340 mM NaCl, 20 mM EDTA, 4mM EGTA, 0.02 % digitonin, 0.05 mg/ml RNAseA, 0.05 mg/m, 2 pg/ml heterologous spike-in DNA) were added to each sample, and mixed with gentle vortexing. Samples were incubated for 10 min at 37°C, then centrifuged for 5 min at 4°C at 16,000 rpm. The tubes were placed on the magnetic stand and the supernatant was transferred to a clean 1.5 ml microcentrifuge tube. DNA extraction was performed utilizing standard phenol chloroform and ethanol precipitation methods as described (Skene et al. 2018). Samples were ethanol precipitated overnight at -20°C. DNA pellets were dissolved in 20 ul 0.1x TE Buffer (1 mM Tris-HCl pH 8, 0.1 mM EDTA). The Qubit High-Sensitivity Assay was used for DNA quantification. Cut&Run libraries were prepared using the KAPA Hyper Prep Kit protocol, with some modifications. The total volume of Cut&Run DNA was used for library construction. For adapter ligation, 5 μl of 3 μM Adapter stock from the KAPA Dual-Indexed Adapter Kit was used. The ligation was incubated at 20°C for 15 min. The library was amplified using the following cycling conditions: 98°C - 45 sec, 98°C -15 sec, 60°C -10 sec, 14 cycles, 72°C for 1 min. After library amplification, the libraries were purified using 50 ul of KAPA Pure Beads and eluted in 20 μl of water. mouse E13.5 ganglionic eminences were dissected, and single cell suspensions were processed for CUT&RUN, mouse E13.5 ganglionic eminences were dissected, and  processed for 4Cseq