Sirt2 ChIP-Seq of Subcortical White Matter

The subcortical WM (SCWM) of WT mice from P12-P15 (reared under Nx and Hx conditions) was dissected and snap frozen. Chromatin was sonicated and immunoprecipitation with anti-Sirt2 antibody (Santa Cruz, sc211033) or IgG negative control was performed using the High Sensitivity ChIP kit from Abcam (Abcam 185913). DNA samples were sent to the Penn State Hershey Genome Sciences and Bioinformatics Facility and Penn State College of Medicine. Libraries were prepared using the NebNext kit for input of <100 ng DNA, according to the manufacturer’s protocol. 50 base pair, paired-end reads were obtained from the NovaSeq 6000 System (Illumina). Reads were mapped to the mouse reference genome (Genome Reference Consortium Mouse Build 38—mm10) using bowtie2 (version 2.4.4)^{82 (link)}, and mitochondrial chromosomes were removed using Samtools (version 1.7)^{83 (link)}. Duplicates were marked with Picard (version 2.26.10) and removed with Samtools. Binding peaks were identified using Macs2 (version 2.2.7.1)^{84 (link)} using an adjusted p value <0.1 as threshold. Sirt2 ChIP-seq data were compared with the RNA-seq data previously published^{39 (link)}.