GSM6124722: Rag1 KO, Batch 1, White Matter; Mus musculus; RNA-Seq
The mice were deeply anesthetized and perfused with cold HBSS between 9am-11am (to decrease circadian fluctuations). Each brain was removed and under a dissection microscope individually micro-dissected; grey matter was isolated from the frontal cortex and white matter form optic tract, medial lemniscus and corpus callosum (attached grey matter and choroid plexus were removed carefully) isolated. We developed and established a microglia isolation protocol that prevents ex-vivo transcription and automatizes the mechanical isolation parts using GentleMacs with the Neural Tissue Dissociation Kit (Papain) (Miltenyi Biotec). We added actinomycin D (Act-D, Sigma, No. A1410) to a final concentration of 45 μM into the dissociation solution and enzyme mix to prevent ex-vivo transcription. To collect enough cells for loading onto the 10x Genomics Chromium chip, two gray matter/ white matter tissue samples were combined into one sample. After tissue dissociation, SYTOX Blue negative cells were sorted into a 2 mL Eppendorf tube with 1 mL RPMI+5% FBS. Sorted cells were centrifuge at 300xg for 10min at 4ºC. Cell pellets were resuspended in 0.04% BSA+PBS catching media at a concentration of 700-900 cells per μl. Single-cell suspensions were loaded onto the Chromium Single Cell Controller using the Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (10X Genomics) chemistry following the manufacturer's instructions. Sample processing and library preparation was performed according to manufacturer instructions using AMPure beads (Beckman Coulter).