GSM7426821: OF2; Mus musculus; RNA-Seq
For CD11b-MACS: Hippocampal cells from C57BL6/N mice were pelleted at 300 x g for 10 minutes at 4°C then resuspended in 90 µL of 0.5% BSA in D-PBS and 10 µL of CD11b (Microglia) MicroBeads (#130-093-634, Miltenyi Biotec) followed by thorough mixing. The cells were then labeled by incubating with the CD11b-beads for 15 minutes at 2-8°C. After incubation, labeled cells were washed in 1 mL of 0.5% BSA in D-PBS and then centrifuged at 300 x g for 10 minutes. Cells were resuspended in 700 µL of 0.5% BSA in D-PBS. 200 µL of the cell suspension was reserved as the CD11b-MACS Input for flow cytometric and RNA-Seq analyses. The remaining 500 µL was processed on the autoMACS Pro Separator (#130-092-545, Miltenyi Biotec). After priming the autoMACS instrument, the sample and collection tubes were placed in a cold Chill5 tube rack (#130-092-951, Miltenyi Biotec), collecting both positive and negative fractions. The double-positive selection (Posseld) program was used to elute highly pure CD11b+ cells. For Cx3cr1-TRAP: Hippocampal tissue was isolated from Cx3cr1-NuTRAP mice of both sexes at 6 and 22 mo (n=5-7/sex/age). The hippocampi from both hemispheres were minced into small pieces and homogenized in 100 µL ice-cold TRAP homogenization buffer (50 mM Tris, pH 7.4; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 1 mg/ml sodium heparin; 1 mM DTT; 100 μg/ml cycloheximide [#C4859-1ML, Millipore Sigma]; 200 units/ml RNaseOUT Recombinant Ribonuclease Inhibitor [#10777019; Thermo Fisher Scientific]; 1× complete EDTA-free Protease Inhibitor Cocktail [#11836170001; Millipore Sigma] with a Kimble pellet pestle mixer [#749540-0000; DWK Life Sciences), as previously described (7, 8). Volume was brought up to 1.5 mL with TRAP homogenization buffer. Homogenates were centrifuged at 12,000 × g for 10 min at 4°C. After centrifugation, 100 μL of the cleared supernatant was saved as TRAP Input. The remaining supernatant was incubated with 5 μg/μL of anti-GFP antibody (ab290; Abcam) at 4°C with end-over-end rotation for 1 hour. A volume of 50 µL per sample of Protein G Dynabeads (#10003D; Thermo Fisher Scientific) was washed three times in 1-mL ice-cold low-salt wash buffer (50 mM Tris, pH 7.5; 12 mM MgCl2; 100 mM KCl; 1% NP-40; 100 μg/ml cycloheximide; 1 mM DTT) and then resuspended in the original volume of low salt wash buffer. 50 µL of washed beads were added to each cleared homogenate and incubated at 4°C with end-over-end rotation overnight. Following overnight incubation, the beads and GFP-bound polysomes were washed three times with 0.5 mL of high-salt wash buffer (50 mM Tris, pH 7.5; 12 mM MgCl2; 300 mM KCl; 1% NP-40; 100 μg/mL cycloheximide; 2 mM DTT) using a magnetic stand for separation. After the last wash, 350 μL of Buffer RLT (Qiagen) was supplemented with 3.5 μl of 2-β mercaptoethanol (#444203, Millipore Sigma) and added to the washed beads. Samples were incubated with mixing on a ThermoMixer (Eppendorf) for 10 minutes at room temperature. The beads were magnetically separated, and the supernatant (Positive Fraction) was transferred to a new tube. RNA was isolated using the RNeasy Mini kit (#74104, QIAGEN) according to the manufacturer's instructions. RNA was quantified with a Nanodrop Onec spectrophotometer (#ND-ONEC-W, Thermo Fisher Scientific), and its quality was assessed by HSRNA ScreenTape (#5067-5579, Agilent Technologies) with a 4150 Tapestation analyzer (#G2992AA, Agilent Technologies). RNA was isolated using the Qiagen Rneasy Mini Kit according to the manufacturer's instructions. Directional RNA-Seq libraries were made according to the manufacturer's protocol from 5-100 ng RNA. Poly-adenylated RNA was captured using NEBNext Poly(A) mRNA Magnetic Isolation Module (#NEBE7490L, New England Biolabs) and then processed using NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L, New England Biolabs) for the creation of cDNA libraries. Library sizing was performed with HSD1000 ScreenTape (#5067-5584, Agilent Technologies) and quantified by Qubit dsDNA HS Assay Kit (#Q32851, ThermoFisher Scientific) on a Qubit 4 Fluorometer (#Q33226, ThermoFisher Scientific). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NovaSeq 6000 system (SP PE50bp, S4 PE150).