Injured_adult_Treated_Mus Musculus
C57BL/6 mice of mixed sex (8 to 10 weeks old, 20-22g) were used in this study, with all procedures approved by the Animal Ethics Committee at CSIR CCMB and adhering to Institutional Animal Ethics Committee (IAEC) guidelines. Mice received anterograde injections of viral vectors at four points relative to bregma (in mm): (x1=+2, y1=0), (x2=+2, y2=+1), (x3=-2, y3=0), and (x4=-2, y4=+1). A total of 2.4 L virus was injected (0.6 L per point), with animals receiving either control (GFP AAV9) alone or a combination of NR2F6 AAV9 and NR2F1 AAV9 (exact titers provided in Supplementary Table 2). One-week post-injection, animals underwent thoracic crush injury as previously described. One week after injury, animals were euthanized, and motor cortices were dissected, flash-frozen on dry ice, and stored at -80C until library preparation. Frozen tissue was homogenized in 2 mL Nuclei Lysis Buffer (Sigma, NUC101) supplemented with RNase inhibitors (10 mg/mL) using a pre-chilled 2 mL dounce homogenizer. Nuclei were pelleted by centrifugation (500 G, 2 min, 4C, low brake and acceleration) and resuspended in 100 L Nuclei Resuspension Buffer. Dissociated nuclei were filtered through a 20 m filter and flow-sorted on a BD FACS Melody using an 80 m nozzle, targeting 4500-5000 events. Sorted nuclei were collected in Nuclei Resuspension Buffer containing 10 RT buffer. Library preparation was performed on the 10 Chromium platform according to the manufacturer's instructions (10 Genomics-Next GEM, Cat # PN1000121). The flow-sorting procedure employed a sequential gating strategy (Sort type: Purity) as before10. Nuclei were first gated by side scatter height (SSC-H) vs. forward scatter height (FSC-H) and side scatter area (SSC-A) vs. FSC-A to separate debris from intact nuclei. Nuclei passing these filters were then gated by SSC-W vs. SSC-H to eliminate potential doublets. Finally, nuclei were gated by levels of fluorescence marker, selecting only the brightest nuclei for collection, aiming for 4500-5000 events for single-nuclei RNA-Seq.