RNA-seq of mouse microglia
The quality of RNA extracted from isolated microglia was analyzed by Bioanalyzer 2100 (Agilent Technologies), RNAs with RNA integrity number >8 were further processed. The TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (500 ng) were poly-A selected and then reverse-transcribed into double-stranded cDNA with Actinomycin added during first-strand synthesis. The cDNA samples were fragmented, end-repaired and adenylated before ligation of TruSeq adapters. The adapters contain the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit (1.0) Fluorometer (Life Technologies, California, USA) and the Bioanalyzer 2100 (Agilent Technologies). The product is a smear with an average fragment size of approximately 360 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 4000 single end 125 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA). Reads were quality-checked with FastQC. For RNAseq data analysis, we used RSEM to perform RNAseq read alignment and expression estimation(Li & Dewey, 2011). As reference we used the GRCm38 genome assembly and the corresponding gene annotations provided by Ensembl. We computed differential expression with the Bioconductor package DESeq2(Love et al, 2014). A gene was considered as differentially expressed by applying a threshold of 0.01 for the p-value and 1 for the log-ratio. Additionally, we filtered away genes that had very low counts. Specifically, we did not consider a gene as expressed if it did not exceed in at least one condition an average read count of 10 in the samples. All clusterings and visualizations were performed with R/Bioconductor (R version 3.2).