GSM9144907: Cut&Tag, SOX9, 1dpi, M; Mus musculus; OTHER
Spinal cords were microdissected and cleared of the meninges. We obtained 5 mm segments of tissue around the injury epicenter, or at the same rostrocaudal level in uninjured animals. Nuclei from WT mice were isolated with the Chromium Nuclei Isolation Kit (10X Genomics, PN-1000493) and sorted on BD FACS Influx sorter with a 100 μm nozzle, based on 7AAD (1h on ice, 1:50) staining. A total of 150,000 nuclei from each sample were used to perform CUT&Tag. Nuclei were incubated overnight at 4°C with Sox9 antibody (1:100 dilution; Sigma AB5535) in antibody buffer. 20% of the nuclei per sample were set aside as secondary-only controls and underwent all other steps in parallel. After washing, nuclei were incubated for 1 hour with guinea pig anti-rabbit secondary antibody (1:50 dilution; Novus Biologicals, NBP1-72763) in dig-wash buffer. After washing, samples were incubated with loaded pA-Tn5 transposase (1:100 dilution; C01070001) and subsequently tagmented for 1 hour at 37°C. After the reaction was stopped, DNA was purified using MinElute Reaction cleanup kit (Qiagen 28204) and directly used for PCR amplification and indexing for 15 cycles using NEBnext High Fidelity master mix (NEB, M0541). Samples were then sequenced on an MGI G99 sequencer using paired-end reads according to manufacturer's protocols.
GEO
GSM9144907_r1