GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3305490

Query DataSets for GSM3305490

Status

Public on Jul 25, 2018

Title

s_HAS_1

Sample type

SRA

Source name

Saline control for Poly(I:C) study

Organism

Mus musculus

Characteristics

tissue: forebrain microglia
protocol: RiboTag

Extracted molecule

total RNA

Extraction protocol

To investigate alterations in the microglial translatome during normal aging, systemic inflammation, amyloidoisis and tauopathy, RNA was isolated from microglia using the RiboTag translational Cre/Lox-based profiling system for microglial transcript isolation based on expression of a hemagluttanin (HA) epitope-tagged core ribosomal protein (RPL22) in a cell type of interest. A tamoxifen inducible Cx3cr1 promoter for Cre and eYFP expression (Cx3cr1-CreERT2-IRES-eYFP was crossed to RiboTag mice to generate double heterozygous Cx3cr1CreERT2-IRES-eYFP/+; Rpl22HA/+ mice (referred to as RiboTag). For comparisons of RiboTag vs cellular isolation strategies at baseline and following LPS injection, microglia were also isolated from the forebrain of PBS perfused tamoxifen injected RiboTag mice using cellular isolation strategies where cells underwent a 20 minute collagenase enzymatic digestion followed by a Percoll gradient for myelin removal and sorting based on staining for CD45 and CD11b (7AAD- CD11b+ CD45lo/int microglial population were sorted to >98% purity). Cells were sorted into Buffer RLT supplemented with 2-β-mercaptoethanol and RNA was isolated using a Qiagen RNeasy microkit with in column DNaseI digestion. For all RiboTag studies, RiboTag mice (Cx3Cr1cre/+; Rpl22HA/+) were injected for 3 consecutive days with 100μg/g tamoxifen and allowed to rest for a week prior to harvesting or systemic challenge followed by harvest. PBS perfused forebrains were immediately sonicated in homogenization buffer (50mM Tris pH 7.4, 100mM KCl, 12mM MgCl2, 1% NP-40) supplemented with 1mM DTT, 1x protease/phosphatase inhibitors, 200U/mL RNAsin, 100 μg/mL cycloheximide, and 1mg/mL heparin. Lysates were centrifuged at 4°C at 10,000g for 10 minutes. Supernatants were incubated with anti-HA antibodies and rotated at 4°C for 4 hours prior to incubation with protein G magnetic beads overnight at 4°C while rotating. Beads were magnetized and non-HA containing supernatants were removed prior to washing with a high salt buffer (50mM Tris, 300mM KCl, 12mM MgCl2, 10% NP-40, 1mM DTT, 100μg/mL cycloheximide). Three washes were conducted prior to magnetizing the beads, removing all supernatants and then releasing HA bound transcripts from the beads using Buffer RLT supplemented with 2-β-mercaptoethanol. RNA was purified using a Qiagen RNeasy microkit followed by in-column DNase I treatment according to manufacturer’s instructions. For LPS and poly I:C studies, animals were injected with 2μg/g LPS or 12μg/g poly I:C intraperitoneally (i.p.), respectively and microglial were isolated 24 hours post systemic challenge. For amyloidosis studies, 9-10 month old male APPswe/PS1ΔE9 (APP/PS1) and littermate controls were used for RNAseq studies. For tauopathy studies, 9-10 month old male Cx3Cr1creER/+; Rpl22HA/+ that were injected previously injected intracerebroventricularly (ICV) with 2.7E+10 viral particles/ventricle and 2uL/ventricle of rAAV1-TauP301L and rAAV1-GFP on postnatal day 0 were used for to isolate RNA. Finally, in the normal aging study, male or female RiboTag mice (Cx3Cr1cre/+; Rpl22HA/+) were harvested at either 3 months (young), 12 months (middle), or 24 months (old) of age for RiboTag mediated RNA isolation.
Samples were amplified for RNAseq by cDNA library preparation using a NuGen Ovation RNA v2 kit (NuGen, San Carlos, CA) according to manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Base calling by CASAVA
Alignment by Tophat2
Gene count by featureCount
Normalization by RPKM
Genome_build: mm10
Supplementary_files_format_and_content: excel file for rpkm values of genes

Submission date

Jul 25, 2018

Last update date

Jul 26, 2018

Contact name

Xuewei Wang

Organization name

Mayo Clinic

Street address

200 1st street

City

Rochester