title: GSM5962078: microglia, Vehicle, day1 (DRN-22642); Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with cold PBS and brains (excluding cerebellum) were dissected and processed into a single cell suspension using the Adult Brain Dissociation Kit (Miltenyi Biotec), according to the manufacturer's protocol. Cells were Fc blocked and stained for flow cytometric analysis with Fixable Viability Stain BV510 to exclude dead cells (BD Biosciences, #564406), CD11b-BV421 (BD Biosciences, #562605), and ACSA-2-PE (Miltenyi Biotec, #130-102-365). Cells were strained through a 100 μm filter before sorting CD11b+ microglia and ACSA-2+ astrocytes on a FACS Aria III (BD Biosciences) with a 100 μm nozzle. 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). Sequencing libraries were prepared with the Clontech SMARTSeq v4 3′ DE (Takara Bio USA, Inc.) kit for cDNA synthesis and the Nextera XT DNA Library Prep Kit (Illumina) for library generation. 1 ng of total RNA per sample was used for reverse transcription followed by 11 cycles of cDNA amplification to produce full length cDNA. cDNA was purified with 0.8X volume of SPRIselect beads (Beckman Coulter), quantified using Bioanalyzer High Sense DNA chip (Agilent), and then normalized to 300 pg of input for library preparation. The tagmentation step simultaneously fragmented and introduced adaptors to the DNA strands, followed by 12 cycles of PCR amplification. Library quantity and quality were assessed with Qubit™ 1X dsDNA HS Assay Kits (Invitrogen) and Bioanalyzer High Sense DNA chip. Libraries were pooled in equimolar ratios for sequencing in two Illumina HiSeq 4000 lanes at the UCSF Center for Advanced Technology.