Bulk RNAseq of AnnexinV+ and AnnexinV- cells from the Mertk-KO corpus callosum at the 4 week cuprizone timepoint

Purpose: Following 4 weeks of 0.2% cuprizone treatment, Mertk-KO mice accumulate dying cells in the corpus callosum (based on cleaved-cas3 staining). These cells are not seen in Mertk-WT animals at the same cuprizone timepoint. The goal of this study is to identify these dying cells in the Mertk-KO corpus callosum Methods: 2 biological replicates (mice) were used. Corpus callosa were dissected from Mertk-KO mice after 4 weeks of 0.2% cuprizone treatment. Tissues were dissected into single cells using Miltenyi Neural Tissue Dissociation Kit (P) and stained with Annexin V. The stained cell suspensions were then FAC sorted into Annexin V+ and Annexin V- populations. RNA from these cells were extracted using QIAGEN RNeasy kit. Results: Bulk RNA-Seq data were analyzed using an in-house pipeline consisting of GSNAP HTSeqGenie. Reads aligning uniquely to exons were counted to each gene, and size-factor normalization was used to calculating nRPKM statistic. Conclusions: Based on the differences of transcriptomic profiles of AnnexinV+ and AnnexinV- cells, we conclude that the dying AnnexinV+ cells are microglia. Overall design: Comparison of dying (AnnexinV+) and living (AnnexinV-) cells in the Mertk-KO corpus callosum after 4 weeks of 0.2% cuprizone treatment.