GSE127543
RNA-seq of microglia isolated from aged mice treated with IgG or anti-CD22.
Sequencing data related to the manuscript entitled, "CD22 blockade restores homeostatic microglial phagocytosis in the aging brain." Overall design: To assess the transcriptional effects of CD22 blockade, we implanted aged mice with osmotic pumps to continuously infuse a CD22 blocking antibody or an IgG control antibody directly into the cerebrospinal fluid for one month. Following one month of continuous infusion, we performed RNA-seq on purified microglia from the hemi-brains of these mice contralateral to the cannulation site to minimize injury-induced confounding factors. Primary mouse microglia were isolated by gentle dounce homogenization of the brain, magentic myelin removal, and FACS-purification of ~20,000 live CD11b+CD45lo cells. Microglia were sorted into RLT Plus buffer (Qiagen) containing beta-mercaptoethanol. RNA was extracted using a RNeasy Micro Plus kit (Qiagen) according the manufacturer's protocol. RNA integrity was assessed on a Bioanalyzer (Agilent), and high quality samples were used for library preparation. cDNA synthesis and amplification was performed using the SmartSeq v4 Ultra-low input kit (Takara), and libraries were tagmented, adaptor ligated, and indexed using the Nextera XT kit (Illumina). After normalization and pooling, libraries were sequenced on a Hiseq 4000 (Illumina) using paired-end 100bp reads. Raw sequencing files were demultiplexed with bcl2fastq, reads were aligned using STAR, the count matrix was generated using SummarizedExperiment, and differential expression analysis was performed using DESeq2 with standard settings.