<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This file contains the mRNA-seq dataset that was used in the unpublished manuscript and was further analyzed with the DEGviewer online software.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).

本文件包含了用于某篇未发表手稿的mRNA测序（mRNA-seq）数据集，该数据集已通过DEGviewer在线软件开展进一步分析。相较于对照组，CTE诱导组的mRNA表达呈现出显著的倍数变化。总RNA浓度采用Quant-IT RiboGreen（货号R11490，Invitrogen）进行定量检测。为评估总RNA完整性，样本使用TapeStation RNA ScreenTape（货号5067-5576，安捷伦科技，德国瓦尔德布隆）进行质检。仅选取RNA完整性数值（RIN）大于7.0的高质量RNA制剂用于RNA文库构建。每个样本取1 μg总RNA，通过Illumina TruSeq Stranded 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）独立完成文库构建。实验流程第一步为：利用结合有poly-T的磁珠富集含poly-A尾的mRNA分子。富集完成后，在高温环境下通过二价阳离子将mRNA片段化为短片段。使用SuperScript II反转录酶（货号18064014；Invitrogen）与随机引物，将断裂后的RNA片段反转录为第一链cDNA。随后借助DNA聚合酶I、RNase H及dUTP完成第二链cDNA的合成。上述cDNA片段依次经过末端修复、添加单碱基“A”尾，随后连接测序接头。纯化产物经PCR富集后得到最终的cDNA文库。文库的定量采用适配Illumina测序平台的KAPA Library Quantification试剂盒，参照qPCR定量方案指南（货号KK4854，Kapa Biosystems，美国马萨诸塞州沃本）完成；文库质量则通过TapeStation D1000 ScreenTape（货号5067-5582，安捷伦科技）进行评估。将带有索引的文库上机至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（2×100 bp）测序。