SMART-seq of Microglia in a Mouse Subarachnoid Hemorrhage Model

AbstractSubarachnoid hemorrhage (SAH) is a devastating form of stroke with high mortality and morbidity. The inflammatory response, particularly the activation of microglia, the resident immune cells of the brain, is a critical contributor to early brain injury and secondary complications following SAH. However, the precise transcriptomic changes in microglia during the acute phase of SAH remain incompletely characterized, in part due to the technical challenge of obtaining sufficient high-quality RNA from these acutely isolated cells. This study employs the SMART-seq2 protocol, a full-length RNA-seq method optimized for low-input samples, to comprehensively map the transcriptome of microglia acutely isolated from the brains of wild-type mice following experimental SAH.Experimental Design and MethodsWe induced SAH in adult wild-type C57BL/6 mice using a standardized prechiasmatic injection model. Sham-operated animals served as controls. At 24 hours post-operation, a critical time point for acute neuroinflammation, microglia were acutely isolated from the whole brain using a combination of mechanical and enzymatic dissociation, followed by CD11b-positive selection. Due to the limited cell yield from this acute isolation protocol, total RNA was extracted and amplified using the SMART-seq2 method to construct sequencing libraries. This approach allows for high-sensitivity, full-length transcript coverage from minute amounts of input RNA. Sequencing was performed on the Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads. The dataset includes a total of 8 samples: 3 biological replicates for the sham group and 5 biological replicates for the SAH group.Data Content and Potential ApplicationsThis dataset provides a detailed and quantitative view of the gene expression changes in microglia during the acute phase of SAH, specifically enabled by a low-input RNA-seq technology. The raw sequencing data (FASTQ files) are deposited here. We anticipate that this resource will be invaluable for:Identifying differentially expressed genes (DEGs) and key signaling pathways involved in the acute microglial response to SAH with high transcript detection sensitivity.Gaining insights into the molecular mechanisms underlying microglia-mediated neuroinflammation and neural damage.Discovering novel transcriptional regulators and potential therapeutic targets for mitigating early brain injury after SAH.Serving as a high-quality reference for studying microglial biology in conditions where cell numbers are limited.Enabling alternative splicing and isoform-level analysis due to the full-length transcript coverage provided by the SMART-seq2 method.

摘要
蛛网膜下腔出血（Subarachnoid hemorrhage, SAH）是一类致死率与致残率均较高的毁灭性卒中亚型。炎症反应，尤其是大脑固有免疫细胞小胶质细胞（microglia）的活化，是SAH后早期脑损伤及继发并发症的关键致病诱因。然而，SAH急性期中小胶质细胞的精确转录组变化尚未得到完全阐释，其部分原因在于从这类急性分离细胞中获取足量高质量RNA存在技术瓶颈。本研究采用针对低起始量样本优化的全长RNA测序技术SMART-seq2，对实验性SAH模型野生型小鼠脑中急性分离的小胶质细胞开展全面转录组图谱绘制。

实验设计与方法
我们采用标准化视交叉前注射模型，在成年野生型C57BL/6小鼠中诱导SAH，以假手术动物作为对照。在术后24小时这一急性神经炎症的关键时间节点，联合机械解离与酶解方法从全脑分离急性小胶质细胞，随后通过CD11b阳性筛选富集目标细胞。鉴于该急性分离方案的细胞产量有限，我们提取总RNA并通过SMART-seq2方法进行扩增以构建测序文库。该技术可从极微量起始RNA中实现高灵敏度的全长转录本覆盖。测序在Illumina NovaSeq 6000平台上完成，生成150 bp双端读段。本数据集共包含8个样本：假手术组3个生物学重复，SAH组5个生物学重复。

数据内容与潜在应用
本数据集借助低起始量RNA测序技术，实现了SAH急性期小胶质细胞基因表达变化的精细定量解析。原始测序数据（FASTQ格式文件）已在此处提交存档。我们预计该资源将在以下方面具有重要应用价值：
1. 以高转录本检测灵敏度，鉴定SAH后急性小胶质细胞应答相关的差异表达基因（differentially expressed genes, DEGs）与关键信号通路；
2. 深入解析小胶质细胞介导的神经炎症及神经损伤的分子机制；
3. 发现可用于缓解SAH后早期脑损伤的新型转录调控因子与潜在治疗靶点；
4. 作为高质量参考数据集，用于研究细胞数量受限条件下的小胶质细胞生物学特性；
5. 依托SMART-seq2提供的全长转录本覆盖度，开展可变剪接及异构体水平的分析。