Treatment of Nr2f6 Cut and Run data
Animals received anterograde injections of AAV9-NR2F6, followed by a Thoracic Crush one week later. One-week post-injury, cortices were dissociated and nuclei extracted as described before. CUT&RUN was performed using the Active Motif CUT&RUN Assay Kit (Catalog No. 53180) following the manufacturer's instructions. Briefly, 50,000 nuclei were used as starting material for each sample. Nuclei were bound to Concanavalin A beads and incubated overnight at 4C with NR2F6 antibody (Thermofisher, PA5-23321). After washing, pAG-MNase was added and activated with CaCl2 for targeted chromatin digestion. DNA was extracted, purified, and used for library preparation with the DNA Library Prep Kit for Illumina (Active Motif Cat. No. 53220). Input samples were prepared similarly, except they were sonicated to yield 400-700 bp fragments and did not undergo antibody binding or pAG-MNase digestion. Libraries were amplified using recommended PCR cycling conditions and cleaned up using a double-sided SPRI bead protocol. Both Input and CUT&RUN samples were sequenced to a depth of 15 million paired-ends each on an Illumina NovaSeq platform.