GSM4976706: CAh12-2_KO; Mus musculus; RNA-Seq
Brain was excised after PBS (Thermo Fisher Scientific) perfusion, minced and digested with 450 U ml-1 collagenase I, 125 U ml−1 collagenase XI, 60 U ml−1 DNase I and 60 U ml−1 hyaluronidase (Sigma) in PBS for 40 min at 37 °C. Samples were passed through a 70-μm cell strainer and mixed with 30% percol layered on-top of 70% percol. The percol gradient was centrifuged at 500g for 30 mins with brake off. The cell fraction was collected and washed with PBS. Microglia were identified as live (7AAD-), CD45+, CD11b+ and sorted using a FACS Aria II cell sorter (BD Biosciences). Microglia were pooled as described above and total RNA was isolated using E.Z.N.A micro elute total RNA kit according to the manufacturer's instructions (Omega Biotek). NEBNext PolyA Directional RNA Library Prep Kit for Illumina