Patz1_h3k27_Cut and Run
Mice received anterograde injections of AAV9-PATZ1, followed by an injury to the thoracic region 7 days post-injection. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) technique was performed using the CUT&RUN DNA-Protein Interaction Assay Kit (Cell Signaling Technology, #86652) according to the manufacturers guidelines. Motor cortices were extracted from the brains of treated mice, and approximately 50,000 nuclei were isolated for the assay. PATZ1 antibody (MyBioSource, MSB9403528) was added to the nuclei bound to Concanavalin A beads, and the mixture was incubated overnight at 4C. DNA at specific targeted regions was cleaved using pAG-MNase, which was activated by CaCl. The resulting DNA fragments were purified and used to produce sequencing libraries using a library preparation kit (Catalogue number :86652S ). Input samples were prepared using a similar protocol, except they were not treated with antibody or pAG-MNase but were instead sonicated (Covaris M220) to produce fragments between 100-600 bp (Peak incident: 75, Duty factor: 10, Cycles/Burst: 200, Treatment time: 75 seconds ). Both CUT&RUN and input libraries were PCR amplified, and sequencing was performed to a depth of 5 million paired-end reads on the Illumina NovaSeq platform.Similarly, motor cortices were isolated from uninjured mice, mice 7 days post-injury, and mice treated with AAV-PATZ1 prior to thoracic injury. CUT&RUN assays were performed using the H3K27ac antibody (Abcam, Catalogue No:ab4729 ) and CTCF antibody (Cell Signalling Technology, Catalogue No:3418 ), following the manufacturers protocol outlined above. The libraries prepared were then amplified, and sequenced to a depth of 15 million paired-end reads.