Discover - Cerebellum

Genome wide methylome and whole proteome profiling was performed on cerebellum samples collected from Ptch1⁺/⁻ mice following neonatal γ irradiation (0 Gy, 0.1 Gy, 2 Gy). Genomic DNA was extracted from frozen cerebellar tissue and analyzed by reduced representation bisulfite sequencing (RRBS). Reads were aligned to the reference genome using Bismark, and methylation levels at CpG sites were quantified. Differential methylation analysis was performed using the methylKit R package. The average methylation at each chromosome and at each annotated transcript are determined on a per-sample basis. For the region-based analysis, the genome is tiled into 1 kilobase regions and significantly differentially methylated tiles are identified (methylation difference > 20%). Proteins were extracted from frozen cerebellar tissue, digested, and analyzed by DIA-PASEF mass spectrometry. Data were processed using Spectronaut against the SwissProt mouse database. Differential protein expressions were defined based on q-value 0.05, at least two unique peptides, and fold change ≥1.5 or ≤0.6.

本研究针对新生期经γ射线照射（照射剂量分别为0 Gy、0.1 Gy、2 Gy）的Ptch1+/-小鼠的小脑组织样本，开展全基因组甲基化组与全蛋白质组谱分析。从冻存小脑组织中提取基因组DNA，采用简化代表性亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）进行分析；使用Bismark工具将测序reads比对至参考基因组，并定量CpG位点的甲基化水平。采用methylKit R包开展差异甲基化分析，以单样本为单位统计每条染色体及每个注释转录本的平均甲基化水平；在区域层面的分析中，将基因组划分为1 kb的连续瓦片区域，筛选甲基化差异幅度超过20%的显著差异甲基化瓦片。从冻存小脑组织中提取蛋白质，经酶解后采用DIA-PASEF质谱进行检测；利用Spectronaut软件结合SwissProt小鼠数据库对质谱数据进行处理。差异蛋白表达的筛选标准为：q值≤0.05、至少包含2条独特肽段，且折叠变化≥1.5或≤0.6。