title: Illumina MiniSeq paired end sequencing; RNA Panel sequencing of isolated microglia of wild-type and TSPO knockout mice treated with diazepam (5mg/kg) or vehicle daily for one week.
design: RNA Panel sequencing of isolated microglia of wild-type and TSPO knockout mice treated with diazepam (5mg/kg) or vehicle daily for one week.
library_construction_protocol: Microglia were immunomagnetically isolated according to the manufacturer's instructions (Miltenyi Biotec, Germany). Briefly, mouse brain cortices were dissected and minced on ice. The minced tissues were enzymatically dissociated using Adult Brain Dissociation Kit (Miltenyi Biotec, Germany) and further mechanically dissociated by the gentleMACS Octo Dissociator (Miltenyi Biotec, Germany). Tissue debris and red blood cells were subsequently removed with multiple rounds of incubation and centrifugation as instructed in the manufacturer's guidelines. The prepared single-cell suspensions were immediately incubated with anti-CD11b (Microglia) MicroBeads (Miltenyi Biotec, Germany) and separated using MACS LS columns (Miltenyi Biotec, Germany) in a magnetic field of a QuadroMACS Separator (Miltenyi Biotec, Germany). Cells in the CD11b+ fraction were collected and used for the subsequent experiments. Total RNA was isolated using RNeasy Plus Micro Kit and automatically processed on a QIAcube Connect system (QIAGEN, Germany).  The library preparation and subsequent sequencing were conducted with TruSeq Targeted RNA Custom Panel Kit, TruSeq Targeted RNA Index Kit A.
attributes.tag: Experimental Factor: compound
attributes.value: diazepam
attributes.tag: Experimental Factor: dose
attributes.value: 5
attributes.tag: Experimental Factor: genotype
attributes.value: TSPO knockout