Next-Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Monocyte-Depleted Brain Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived brain transcriptome profiling (RNA-seq) to reveal the difference of cellular pathways in adult brains of wild type (WT) and monocyte-depleted (CCR2-DTR) mice 8 days post infection of Trichinella spiralis. Methods: Illumina compatible RNAseq library was prepared using NEB next ultra RNAseq library preparation kit. The sequencing of the cDNA libraries was performed on the Illumina NextSeq platform (Illumina, San Diego, CA) using the high output 1X75 cycles configuration. CLC Genomics Workbench 11.0.1 version (http://www.clcbio.com/products/clc-genomics-workbench/ ; Qiagen) was used for RNA-seq analysis. De-multiplexed fastq files from RNA-Seq libraries are imported into the CLC software. Bases with low quality were trimmed and reads are mapped to reference genome Mus musculus genome GRCm38. The reference genome sequence and annotation files were downloaded from ENSEMBLE, release.92 (Mus_musculus.GRCm38.92.fa, and Mus_musculus.GRCm38.92.gtf). The aligned reads were obtained using the RNA-Seq Analysis Tool of CLC Genomics Workbench. Statistical analysis of differentially expressed genes is carried out based on a negative binomial model using a tool in CLC Genomic Workbench. The reference genome sequence and annotation files were downloaded from ENSEMBL, release.92 (Mus_musculus.GRCm38.92.fa, and Mus_musculus.GRCm38.92.gtf). The aligned reads were obtained using the RNA-Seq Analysis Tool of CLC Genomics Workbench. Results: Using an optimized data analysis workflow, we mapped over 50 million sequence reads per sample to the mouse genome (GRCm38) and found that Trichinella-infected mice depleted of monocytes presented with significantly increased expression of proinflammatory genes compared to controls. Overall design: Brain mRNA profiles of adult wild type (WT) and monocyte-depleted (CCR2-DTR) mice 8 days post infection of Trichinella spiralis.