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Sample GSM3744996 Query DataSets for GSM3744996
Status Public on Dec 21, 2019
Title Pooled microglia cells from two Trem2 +/+ animals after 12 weeks on control diet
Sample type SRA
 
Source name WT microglia, control diet
Organism Mus musculus
Characteristics Sex: male
age: 12-14 months
genotype: Trem2 +/+
diet: control
tissue: brain
cell_type: microglia
timepoint: 12 weeks
Treatment protocol Trem2-WT, heterozyogus knockout, and homozygous knockout mice were fed a control or 0.2% cuprizone diet for 12 weeks.
Growth protocol Mice were housed on a 12hr/12hr light dark cycle. All mouse husbandry and experimental procedures were approved by Denali's Institutional Animal Care and Use Committee.
Extracted molecule total RNA
Extraction protocol Mice were perfused with PBS, brains were removed and dissociated with the Adult Brain Dissociation Kit (Miltenyi). 30,000 live CD11b+ CD45low microglia were sorted from each hemibrain and two hemibrains per condition were pooled into PBS + 0.5% BSA to generate 4 total sequencing groups. Microglia were counted and diluted to 500,000 cells/mL in 70μL and viability was verified to be >70%.
Single cell libraries were barcoded and prepared using the Chromium Single Cell 3' Library Kit with v2 chemistry (10X Genomics, product #120267) with a Chromium Controller (10X Genomics) at the Stanford Functional Genomics Facility. The libraries were sequenced using a NovaSeq S4 (Illumina) at the UCSF Center for Advanced Technology.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description WT_untx
Data processing The fastq files from each dataset were individually processed using Cell Ranger (v2.1.1). The "raw_gene_bc_matrices" directories from these runs served as the starting point for all subsequent quality control, filtering, and downstream analyses. These steps were performed using a combination of the DropletUtils, scater (McCarthy et al., 2017) and scran (Lun et al., 2016) Bioconductor (v3.8) packages. Experiments were independently quality controlled and filtered by removing low quality droplets. First, droplets containing only ambient levels of RNA were removed. We then calculate per cell: (i) the number of UMIs; (ii) number of genes detected; (iii) fraction of reads that map to ribosomal proteins; and (iv) fraction of reads that map to the mitochondrial genome. Cells that are outliers in any of these metrics (> 2-3 median absolute deviations too low for (i) and (ii), too high for (iii) and (iv)) are removed. Read counts were normalized using a deconvolution strategy for scaling normalization of sparse count data (Lun et al., 2016). All downstream analyses were perform using the 4,895 genes that were identified with positive components of biological variance (i.e., higher variance than the assumed technical Poisson noise) as implemented in the "decomposeVar" method in the scran package.
Genome_build: mm10
Supplementary_files_format_and_content: three csv-delimited text files with 1) the matrix of raw quantitation results for each cell, 2) cell annotations and 3) gene annotations, respectively (more details in the READ.txt)
 
Submission date May 02, 2019
Last update date Dec 21, 2019
Contact name Thomas Sandmann
E-mail(s) genomics@dnli.com, sandmann@dnli.com
Organization name Denali Therapeutics
Street address 161 Oyster Point Blvd
City South San Francisco
State/province California
ZIP/Postal code 94080
Country USA
 
Platform ID GPL24247
Series (2)
GSE130626 TREM2 regulates microglial cholesterol metabolism upon chronic phagocytic challenge [single-cell RNA-Seq]
GSE130627 TREM2 regulates microglial cholesterol metabolism upon chronic phagocytic challenge
Relations
BioSample SAMN11565677
SRA SRX5781924

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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