GSE135230
Cell Autonomous and non-cell autonomous effects of neuronal Bin1 loss in vivo
Purpose: investigate the impact of neuronal Bin1 Loss on surrounding microglia transcriptome. Methods: mRNA profile of  microglia cells FACS-sorted from brains of mice homozygous and heterozygous conditional knock-out for Bin1 in excitatory neurons  (B6.129S6-Bin1tm2Gcp/J (Bin1flox/flox), <FVB/N-Tg(Thy1-cre)1Vln/J> (Thy1-Cre) ) and Bin1-expressing littermates at 3 month of age had been generated from 10ng of total RNA using SMARTer Ultra Low Input RNA kit v4 (Clontech).  The cDNA was amplified by 8 PCR cycles, followed by QC analysis on BioAnalyzer 2100 (Agilent).  Sequence libraries were produced from 150pg of cDNA using Nextera XT DNA library kit (Illumina), cleaned up with AMPure XP beads, and QC checked with Caliper LabChip GX.  Single-end sequencing data were generated on an Illumina Illumina HiSeq 2500, at a depth of 30 million reads per sample, with read length 50bp.The reads were aligned with the OmicSoft OSA4 to the mouse genome (mm10) and the Ensembl.R84 gene model. Gene counts were estimated using RSEM.  Two samples were removed (one sample from WT_Female cohort and one samples from Heterozygote_Male cohort) due to low fraction of uniquely mapped single reads and higher than expected duplication.  Of note, these samples with poor technical QC metrics were also clear outliers in PCA.  Normalization and differential expression analysis were carried out with the Bioconductor package DESeq2. Differentially expressed genes (DEGs) were defined as having adjpval < 0.05, and |FC| > 1.5. Results: Microglia from homozygous Bin1-cKO mice showed 265 (|FC| > 1.5, FDR <0.05) statistically significant Differentially Expressed Genes (DEGs) in comparison to WT mice. Interestingly, microglia from heterozygous Bin1-cKO mice showed no statistically significant (FDR <0.05) DEGs in comparison to WT mice. Conclusion: neuronal disfunction resulting from Bin1 loss alters gene expression of the surrounding microglia. Overall design: 3 month-old mice: Bin1flox/flox X Thy1-Cre+ (Homo-cKO) male mice =5, female mice =5;  Bin1flox/wt Thy1-Cre+ (Het-cKO) male mice = 4, female mice =4; Bin1flox/wt Thy1-Cre- (WT) male mice = 4, female mice =3