|
| Status |
Public on Jul 04, 2024 |
| Title |
MG424DMSOCTL |
| Sample type |
SRA |
| |
|
| Source name |
Primary microglia
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: microglia
sample name: 424
primary treatment: DMSO
secondary treatment: CTL
|
| 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 37C 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
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 |
SAMN20086185 |
| SRA |
SRX11365945 |
