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Sample GSM5424925 Query DataSets for GSM5424925
Status Public on Jul 04, 2024
Title MG524DMSOLPS
Sample type SRA
 
Source name Primary microglia
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: microglia
sample name: 524
primary treatment: DMSO
secondary treatment: LPS
Treatment protocol Microglia were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20uM)(Sigma-Aldrich, C2759), RNA polymerase I Inhibitor 2, CX-5461 (100nM)(EMD Millipore, 509265), or human TGFb1 recombinant protein (10ng/mL)(ThermoFisher Scientific, PHG9204) for 24 hours followed by addition of lipopolysaccharides (LPS, 200nM) from E. coli (Sigma-Aldrich, L2018) for 8 hours and ATP (10nM) for 30 minutes.
Growth protocol Mixed glial cultures were generated from p1 male pups. Cortices and hippocampi were isolated from the pups, and meninges were removed. The tissue was broken up by repeatedly pipetting up and down, followed by dissociation with Trypsin. Single cell suspensions were plated on poly-L-lysine coated flasks in DMEM supplemented with 10% FBS and antibiotic mix (Penicillin/Streptomycin/Amphotericin B) and incubated at 37C in 5% CO2. Media was changed after 4 days to remove debris. Microglia were isolated after 14 days by agitating the plates to dislodge microglia, then transferring the supernatant of each sample evenly amongst 4 wells of a 24 well plate. Microglia were incubated for 16 hours followed by replacement with serum free macrophage media supplemented with M-CSF (10ng/mL)(Peprotech, 315-02) and antibiotic mix.
Extracted molecule polyA RNA
Extraction protocol Tri Reagent was added to the culture plate, and the plates were shaken for 5 minutes. RNA was isolated according to manufacturer’s instructions.
5ng of total RNA was reverse transcribed with SuperScript II (Thermo Fisher) using an oligo-dT RT primer with a PCR binding site and a template switching oligonucleotide with a homotypic PCR binding site. Betaine and MgCl2 were added to enhance the reaction. After reverse transcription, whole transcriptome amplification by PCR was performed for 12 cycles using HiFi HotStart ReadyMix (KAPA) with a PCR primer. The PCR reaction was cleaned up with Ampure XP beads. The amplified DNA was diluted to a concentration of 0.5ng/ul, and subjected to tagmentation with the Illumina Nextera XT kit. Each sample was PCR amplified with a unique set of Nextera indices.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description mginvitrosamples.csv
DMSOLPS_vs_CCCPLPS-ExpDiff.csv
DMSOCTL_vs_DMSOLPS-ExpDiff.csv
Data processing FASTQ reads were pseudo aligned to the mouse transcriptome (GRCm39 cDNA from Ensembl) using kallisto with default parameters.
Differential expression analysis was performed using DeSeq2 using the Wald significance test.
Genome_build: mm10 (GRCm39)
Supplementary_files_format_and_content: Kallisto abundance estimates. Differential expression results and abundance estimates from DeSEQ. Both files types are .csv.
 
Submission date Jul 06, 2021
Last update date Jul 04, 2024
Contact name Jeremy Michael Shea
Organization name University of California, San Francisco
Department Anatomy
Lab Villeda
Street address 513 Parnassus Avenue
City San Francisco
State/province CA
ZIP/Postal code 94143
Country USA
 
Platform ID GPL24247
Series (1)
GSE179611 Microglia aging progresses through functional intermediate steps II
Relations
BioSample SAMN20086181
SRA SRX11365949

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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