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Sample GSM7426817

Query DataSets for GSM7426817

Status

Public on Aug 18, 2023

Title

YM2

Sample type

SRA

Source name

hippocampus

Organism

Mus musculus

Characteristics

tissue: hippocampus
Sex: male
cell type: microglia
genotype: C57BL6/N
age: 5-6 months-old
isolation technique: CD11b-MACS

Extracted molecule

polyA RNA

Extraction protocol

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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

CD11b_cpm_081722.xlsx

Data processing

After sequencing, reads were aligned against the mm10 transcriptome and genome (UCSC) in StrandNGS software (v4.0; Strand Life Sciences). Parameters for alignment and filtering included: adapter trimming, fixed 2-bp trim from 5′ and 2 bp from 3′ ends, a maximum number of one novel splice allowed per read, a minimum of 90% identity with the reference sequence, a maximum 5% gap, and trimming of 3′ ends with Q < 30. Alignment was performed directionally with Read 1 aligned in reverse and Read 2 in the forward orientation, as previously (8).
Binary alignment map (BAM) files were exported from StrandNGS software for further analysis. The Rsubread package (13) was used to quantify the read counts using the featureCount command. Normalization and differential expression calling was then performed using edgeR (14) with the quasi-likelihood pipeline (15). Differentially expressed genes (DEGs) (GLM QLF<0.1, |FC|>1.25) were identified for each of the comparisons (YF v YM, OF v OM, OF v YF, OM v YM) as well as the interaction term.
DEGs were uploaded to Ingenuity Pathway Analysis software (IPA; Qiagen) to assess the biological significance of differential expression. Heatmaps of DEGs and pathway activation z-scores were made using Morpheus software (https://software.broadinstitute.org/morpheus).
Volcano plots were made in R using the EnhancedVolcano package. Upset plots were created using the UpSetR package. Gene set enrichment analysis (GSEA) was run using GSEA v4.1.0 (16). For the differential transcript analysis of the Cdkn2a gene, StrandNGS software was used for quantitation.
Assembly: Mm10
Supplementary files format and content: Xlsx files containg the gene expression (CPM) for all detected genes in each sample

Submission date

May 25, 2023

Last update date

Aug 18, 2023

Contact name

Michael Stout

E-mail(s)

[email protected]

Organization name

Oklahoma Medical Research Foundation

Department

Aging and Metabolism