<b>Bulk RNA Sequencing</b><b> (Mouse Brain)</b>

This file contains the mRNA-seq dataset that was used in the unpublished manuscript and was further analyzed with the DEGviewer online software. The dataset shows significant mRNA fold changes when comparing CTE-induced groups with the control group.
Total RNA concentration was calculated using Quant-IT RiboGreen (cat no. R11490, Invitrogen). To assess the integrity of the total RNA, samples were run on a TapeStation RNA screentape (cat no. 5067-5576, Agilent Technologies, Waldbronn, Germany). Only high-quality RNA preparations (RIN greater than 7.0) were used for the RNA library construction. A library was independently prepared with 1ug of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (cat no. 20020595, Illumina, Inc., San Diego, CA, USA). The first step in the workflow involves purifying the poly‐A containing mRNA molecules using poly‐T‐attached magnetic beads. Following purification, mRNA was fragmented into small pieces using divalent cations at elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using SuperScript II reverse transcriptase (cat no. 18064014; Invitrogen) and random primers. This was followed by synthesis of second-strand cDNA using DNA Polymerase I, RNase H, and dUTP. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products were purified and enriched using PCR to create a final cDNA library. The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms, according to the qPCR Quantification Protocol Guide (cat no. KK4854, Kapa Biosystems, Woburn, MA, USA) and qualified using a TapeStation D1000 ScreenTape (cat no. 5067-5582, Agilent Technologies). Indexed libraries were then submitted to an Illumina NovaSeq6000 (Illumina, Inc., San Diego, CA, USA) and paired-end (2×100 bp) sequencing was performed by Macrogen, Inc. (Daejeon, South Korea).

本文件包含未发表手稿中所使用的mRNA测序（mRNA-seq）数据集，该数据集后续通过DEGviewer在线软件开展了进一步分析。在CTE诱导组与对照组的比较中，该数据集展现出显著的mRNA差异倍数。
总RNA浓度采用Quant-IT RiboGreen（货号：R11490，Invitrogen公司）进行定量。为评估总RNA的完整性，将样本通过TapeStation RNA ScreenTape（货号：5067-5576，安捷伦科技公司，德国瓦尔德布龙）进行检测。仅采用RNA完整性数值（RNA Integrity Number, RIN）大于7.0的高质量RNA样本开展RNA文库构建。每个样本取1 μg总RNA，使用Illumina TruSeq链特异性mRNA样本制备试剂盒（货号：20020595，Illumina公司，美国加利福尼亚州圣地亚哥）独立构建文库。该建库流程的第一步为：使用结合有poly(T)的磁珠富集含有poly(A)尾的mRNA分子。富集完成后，在高温条件下通过二价阳离子将mRNA片段化为小片段。使用SuperScript II反转录酶（货号：18064014，Invitrogen公司）与随机引物，将断裂后的RNA片段反转录为第一链cDNA。随后使用DNA聚合酶I、RNase H及dUTP合成第二链cDNA。随后对这些cDNA片段进行末端修复、添加单碱基“A”尾，最后连接测序接头。对产物进行纯化并通过聚合酶链式反应（PCR）富集，得到最终的cDNA文库。参照实时定量聚合酶链式反应（qPCR）定量实验指南，使用适配Illumina测序平台的KAPA文库定量试剂盒（货号：KK4854，Kapa Biosystems公司，美国马萨诸塞州沃本）对文库进行定量，并通过TapeStation D1000 ScreenTape（货号：5067-5582，安捷伦科技公司）进行文库质量评估。随后将带索引的文库上机至Illumina NovaSeq6000测序平台（Illumina公司，美国加利福尼亚州圣地亚哥），由韩国大田的Macrogen公司完成双端测序（2×100 bp）。