title: GSM5748046: wildtype microbead-injected retina 2; Mus musculus; RNA-Seq
library_construction_protocol: Microglia isolation was performed according to previously described protocols. Briefly, mice were euthanized using CO2, eyes removed, and retinas dissected. PAIRED-cell suspensions were prepared and centrifuged over a 37%/70% discontinuous Percoll gradient (GE Healthcare), and mononuclear cells were isolated from the interface. To distinguish resident microglia from recruited myeloid cells, we used a monoclonal antibody that recognizes Fcrls, which is expressed on microglia but not on infiltrating myeloid cells. Isolated cells were stained with anti-Fcrls [clone 4G11, 3 µg ml-1, Butovsky lab], CD11b-PeCy7 [clone M1/70, BD Biosciences, 2 µg ml-1] and Ly6c-PerCP/Cy5.5 [clone HK1.4, BioLegend, 2 µg ml-1] antibodies to specifically sort resident microglia as CD11b+, Ly6c–, and Fcrls+ cells. 100 to 600 cells were collected from each retina, normalized to 100 to 300 cells per 5 µl of TCL buffer, and submitted for RNAseq. RNA libraries were prepared for sequencing using standard Illumina protocols