GSM4793367: DRN-18455; Mus musculus; RNA-Seq
Mice were perfused with cold PBS and cortical and hippocampal tissues were dissected and dissociated into a single cell suspension using the Adult Brain Dissociation Kit (Miltenyi Biotec 130-107-677), according to the manufacturer's protocol. Cell suspensions were stained with the following antibodies in FACS buffer (1% fatty acid-free BSA + 1mM EDTA in PBS) for 25 minutes on ice: Fixable Viability Stain BV510 (BD Biosciences, 564406), CD11b-BV421 (BioLegend 101251), CD45-APC (BD Biosciences, 559864), ACSA-2-PE (Miltenyi Biotec, 130-102-365), Mouse Fc blocker (anti-mouse CD16/32, BioLegend 101320). Cells were washed twice with FACS buffer and strained through a 100 μm filter before sorting CD11b+ microglia on a FACS Aria III (BD Biosciences) with a 100 μm nozzle. For bulk RNAseq analysis, cells were sorted directly into freshly prepared RLT-plus buffer (Qiagen) containing betamercaptoethanol. Live cells were sorted directly into RLT-plus buffer (Qiagen) with 1:100 betamercaptoethanol. RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) and resuspended in 14 μL nuclease-free water. RNA quantity and quality were assessed with a RNA 6000 Pico chip (Agilent) on a 2100 Bioanalyzer (Agilent). cDNA synthesis was performed with the Clontech SMARTSeq v4 3′ DE kit (Takara Bio USA, Inc. 635040) kit. Two 3' tag RNA-seq libraries were generated per mouse, each consisting of 200 microglia cells isolated by FACS and dispensed directly into 11.5 microliters of 1X Reaction Buffer. Reverse transcription was followed by 15 cycles of cDNA amplification. cDNA was purified with 0.8X volume of SPRIselect beads (Beckman Coulter B23318), quantified on a Bioanalyzer 2100 System with a High Sensitivity DNA chip (Agilent 5067-4626). 300 pg of DNA from each sample was used as input for library preparation with the Nextera XT DNA Library Prep Kit (Illumina FC-131-1096). Fragmentation and adaptors insertion were performed by tagmentation, followed by 12 cycles of PCR amplification. The final libraries were purified using 0.6X SPRIselect beads. Library quantity and quality were assessed with Qubit 4 Fluorometer with the 1X dsDNA HS Assay Kit (Invitrogen Q33231) and on a Bioanalyzer 2100 System with a High Sensitivity DNA chip. 75 bp single end reads were generated on an Illumina NextSeq 500 sequencer by SeqMatic (Fremont, CA, USA)