title: H3K9me3 CUT&RUN of mus musculus: P13 mut cerebellum
design: Between 200,000-500,000 mouse cerebellar nuclei were used in each CUT&RUN experiment. Nuclei were bound to Concanavalin A-coated magnetic beads. Following bead binding, the supernatant was discarded, and the bead-nuclei slurry was resuspended in 75 L of antibody buffer (wash buffer supplemented with 0.005% digitonin and 2 mM EDTA) containing primary antibodies at a concentration of 1:75. Antibody incubation was carriedout overnight at 4C on a nutator. Samples were washed with cold cell permeabilization buffer (Wash buffer with 0.005% digitonin) to remove unbound antibody and then incubated with 1X Protein A/G-MNase in cell permeabilization buffer for 15 minutes at room temperature on a shaker. Samples were washed with cold cell permeabilization buffer and then resuspended in 50 L cell permeabilization buffer containing 2 mM CaCl 2 . Samples were digested for 2 hours at 4C on a nutator. The digestion reactions were quenched with 33 L of STOP buffer (340 mM NaCl, 20 mM EDTA pH 8, 4 mM EGTA pH 7.7, RNase A 0.05 mg/mL, Glycogen 0.05 mg/mL). Samples were then incubated for 10 minutes at 37C. Library preparation for CUT&amp;RUN experiments was performed according to themanufacturers specifications using SPRI selected beads. The concentration of DNA yielded from CUT&RUN was measured on a Qubit device and between 25 and 50 ng of input DNA was used for library preparation, with the amount of input DNA kept constant for all samples using the same antibody. 14 PCR cycles were used in the final amplification with Illumina-compatible adaptors, universal i5 primer and barcoded i7 primers. After measuring the concentration of the amplified product on Nanodrop, all samples were diluted to the same concentration for a given antibody, ranging from 10 ng/uL to 40 ng/uL. Tapestation was performed to determine the precise concentration and fragment distribution range and to ensure there were not significant amount adaptor dimers. Samples were pooled for sequencing 100 bp reads in paired- end configuration on an Illumina NovaSeq X Plus platform at the UCSD Institute for Genomic Medicine.
identifiers.namespace: SUB15172412
identifiers.id: CJF25_index4