title: GSM2565731: EtOH 1; Mus musculus; RNA-Seq
library_construction_protocol: Adolescent mice (age specified in column H) with early life exposure to either saline (control) or ethanol (EtOH) were euthanized by intraperitoneal injection of euthasol and transcardially perfused with ice cold 0.15M phosphate buffer. Each brain was removed and bilateral visual cortex was immediately dissected in ice cold, degassed FACS buffer = 0.5% bovine serum albumin (Sigma A2153) in 1X PBS (Invitrogen 20012-027). Tissue was kept on ice throughout. After dounce homogenization, the tissue with passed through a 70µm filter and centrifuged (1200 RPM, 7 min. 4°C).  Supernatant was aspirated from pellets, which were prepared for magnetic labeling with Myelin Removal Beads II (Miltenyi 130-0960733) according to the manufacturer’s instructions.  Magnetic columns (Miltenyi 130-096-733) were primed with FACS buffer and samples applied to the columns through a clean 70µm filter. After centrifugation of column flow-through and gentle supernatant removal, myelin depleted samples were incubated in Fc block (BioLegend 101320) for 15 minutes at 4°C.   Cells were incubated with CD11b-AlexaFluor 488 (BD Pharmingen 557672) and CD45-APC (BD Pharmingen 561018) for 30 minutes in the dark at 4°C.  The following compensation controls were also freshly prepared in conjunction with experimental samples: 488 bead control, APC bead control (eBiosciences 01-111-42), unstained cells, and triton-X treated cells (propidium iodide (PI) dead cell control). An extra non-experimental fully stained cell sample was included to check voltage settings prior to running experimental samples on an 18 color FACS Aria II flow cytometer. Prior to sorting, all samples and controls were re-suspended in a final FACS buffer volume of 300µL.  PI was added to the samples and appropriate controls 1 hour prior to cell sorting.  For immediate cell lysis and preservation of RNA, microglia (PI negative, CD11b positive, CD45 low) were sorted directly into 1.5mL microcentrifuge tubes (Axygen 311-08-051) containing 600µL Buffer RLT PLUS (Qiagen, 1053393) and 6µL β-mercaptoethanol at 4°C, prepared just prior to sorting.  Experimental samples were not pooled: individual samples represent microglia from bilateral visual cortex from one animal.  The number of CD11b positive CD45 low cells collected (considered the number of microglia collected) is listed in column K above. Total RNA was isolated using the RNeasy Micro Kit (Qiagen). RNA Integrity Number (RIN) was 9.53 ± 0.13 and RNA concentration was 220 ± 17 pg/µL (mean ± SEM) across the 9 samples.  1ng of total RNA was preamplified with the SMARTer Ultra Low Input kit v2 (Clontech). cDNA quality was determined using a Qubit Flourometer (Life Technologies) and Agilent 220 TapeStation. Illumina compatible sequencing libraries were generated from 5ng cDNA with the NexteraXT library prep kit (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM2565731