|
| Status |
Public on Nov 25, 2019 |
| Title |
1yr-Het-12M |
| Sample type |
SRA |
| |
|
| Source name |
Microglia
|
| Organism |
Mus musculus |
| Characteristics |
cx3cr1 genotype: Het
age: 1 year
Sex: M
cell type: Microglia
treatment: None/naïve
|
| Treatment protocol |
Mice were deeply anesthetized with 10 mg/kg ketamine, 30 mg/kg xylazine until they failed to respond to toe pinch and transcardially perfused with cold phosphate-buffered saline (PBS). Mice for the LPS study were euthanized by CO2 asphyxiation, peritoneal cells were isolated by flushing the peritoneal cavity with cold PBS, and then perfused. In all cases, the brain was then isolated and stored in PBS on ice until processing.
|
| Growth protocol |
All mice used in the current manuscript are littermate controls derived from Cx3cr1+/eGFP crosses. The mice were maintained on a normal diet in a 12-hour light/dark cycle. Approximately five mice were used per group (per sex, age, genotype and treatment).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Brains were finely minced with a razor blade and tissues were dissociated to single cell suspension with Accutase on ice for 30 min. Cell suspensins were resuspended with 33% Percoll and centrifuged for 15 min at 800g (no brake). Cells were washed, stained and subjected to sorting for CD11b(+)CD45(lo) cells. RNA was isolated from sorted microglia using Qiagen Universal AllPrep kit (Qiagen 80004) according to the manufacturer’s instructions.
cDNA for RNA-Seq libraries was prepared using 10 ng of RNA using the SmartSeq v4 Ultra Low RNA Input Library prep protocol (Clontech 634889) according to the manufacturer’s instruction.
The cDNA (250 ng starting material) was then tagged and fragmented with the NexteraXT DNA Library Preparation kit (Illumina FC-131-1024) according to the manufacturer’s instructions. Quality of amplified cDNA and fragmented libraries was assessed on a Bioanalyzer with the High Sensitivity DNA chips (Agilent 5067-4627). Tagged libraries were pooled (8 nM total DNA) and sequenced on an Illumina HiSeq2500 sequencer, with target depth of 20 million of 50 bp paired end reads
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Raw reads were aligned to mouse genome build mm10 using STAR version 2.5.2a and transcripts quantified with RSEM version 1.2.26
Genome_build: mm10
Supplementary_files_format_and_content: tab delimited text; gene level count table - *_genes.estcount_table.txt; gene level FPKM table - *_genes.fpkm_table.txt; gene level TPM table - *_genes.tpm_table.txt
|
| |
|
| Submission date |
May 28, 2019 |
| Last update date |
Nov 25, 2019 |
| Contact name |
Stefka Gyoneva |
| E-mail(s) |
Stefka.gyoneva@biogen.com
|
| Phone |
617-914-6196
|
| Organization name |
Biogen, Inc
|
| Street address |
225 Binney St.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE131869 |
Cx3cr1-deficient microglia exhibit a premature aging transcriptome |
|
| Relations |
| BioSample |
SAMN11876733 |
| SRA |
SRX5912317 |
