Tau-P301S sorted neurons and astrocytes

Mice of indicated genotypes were perfused and their brains dissected and dissociated. Cells were fixed, immunolabeled and FACS sorted. RNA was extracted from neuron, astrocyte, and microglial cell populations. Typical RIN=4-5 for neurons, 6-8 for astrocytes, and 5-7 for microglia. Typical RNA yields 200-300ng for neurons, ~20ng for microglia, and ~5ng for astrocytes. cDNA was generated from up to 25 ng of total RNA using Nugen’s RNA-Seq method for low-input RNA samples, Ovation RNA-Seq System V2 (NuGEN). (Per manufacturers instructions, total RNA was neither depleted of rRNA nor polyA-selected.) 1 μg of sheared cDNA was taken into further processing, starting at end repair step, using Illumina’s TruSeq RNA Sample Preparation Kit v2 (Illumina). The SAMPLE_ID sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0025262 NeuN+ neurons, Gfap+ astrocytes, and Cd11b+ myeloid cells were sorted from 6 month old Tau-P301S transgenic mice or non-transgenic littermates. Microglia from the same experiment in GSE93180.

将携带指定基因型的小鼠进行灌流处理，随后取出脑组织并解离。对解离所得的细胞进行固定、免疫标记，随后通过荧光激活细胞分选（FACS, Fluorescence-Activated Cell Sorting）完成细胞分群。从神经元、星形胶质细胞和小胶质细胞群中提取总RNA。样本的典型RNA完整性数（RIN, RNA Integrity Number）为：神经元4~5，星形胶质细胞6~8，小胶质细胞5~7。典型RNA得率为：神经元200~300ng，小胶质细胞约20ng，星形胶质细胞约5ng。使用Nugen公司针对低起始量RNA样本的Ovation RNA-Seq系统V2（NuGEN），以最多25 ng总RNA为模板合成互补DNA（cDNA, Complementary DNA）。（按照制造商说明书操作，总RNA未进行核糖体RNA（rRNA）去除或polyA富集筛选。）取1 μg经片段化处理的cDNA，以Illumina公司的TruSeq RNA样本制备试剂盒v2（Illumina）为工具，从末端修复步骤开始开展后续文库构建流程。SAMPLE_ID为基因泰克（Genentech）内部使用的样本标识符。该项目在基因泰克ExpressionPlot数据库中的编号为PRJ0025262。从6月龄Tau-P301S转基因小鼠及其非转基因同窝仔鼠中分选得到NeuN（Neuronal Nuclei）阳性神经元、胶质纤维酸性蛋白（GFAP, Glial Fibrillary Acidic Protein）阳性星形胶质细胞以及CD11b阳性髓系细胞。本实验的小胶质细胞相关数据已被收录于GSE93180数据集。