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Sample GSM3412003 Query DataSets for GSM3412003
Status Public on Feb 13, 2019
Title P212_A24
Sample type SRA
Source name WT_midbrain_embryonal
Organism Mus musculus
Characteristics tissue: midbrain
age: embryonal
replicate_id: WT_midbrain_embryonal_4
strain: CD1
Extracted molecule total RNA
Extraction protocol After transcardial perfusion with PBS, brains were roughly minced and homogenized with a potter in HBSS containing 15 mM HEPES buffer and 0.54 % Glucose. Whole-brain homogenate was separated by 70/37/30 % layered Percoll gradient centrifugation at 800 g for 30 min at 4 °C (no brake). The CNS macrophages containing interphase was then collected and washed once with PBS containing 2% FCS and 10mM EDTA before staining. Cells were stained with primary antibodies directed against CD11b (M1/70, BioLegend), CD45 (30-F11, BD Biosciences), Ly6C (AL-21, BD Biosciences) and Ly6G (1A8, BD Biosciences) for 20 min, and CD206 (C068C2, BioLegend) for 45 min at 4 °C. After washing, cells were analyd using a MoFlo Astrios (Beckman Coulter). microglia were FACS-sorted from six different CNS regions of healthy and diseased brains (see gating strategy shown in supplementary Fig.2) into a 384-well plate containing a lysis buffer.
Single cell RNAseq libraries were generated following the published SmartSeq2 protocol (Picelli et al. Nature Metods 2013)
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description geosubmission_counts.csv
Data processing Individual Fastq files were aligned to the mouse Genome (Gencode M11 / GRCm38) using the STAR aligner version 2.5.2b with parameters STAR --runThreadN <cpu> --genomeDir <index> --genomeLoad LoadAndKeep --limitBAMsortRAM 20000000000 --readFilesIn <fastq> --readFilesCommand zcat --outFileNamePrefix <sampleID> --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical --outSAMtype BAM SortedByCoordinate
Gene counts were obtained from BAM files using featureCounts v1.6.2 with parameters -a <Gencode_vM11.gtf> -G <GRCm38.primary_assembly.genome.fa> -o counts.txt -F GTF -R -g gene_id -t exon -T 10 –largestOverlap <bamfiles>
single cell RNAseq analysis and clustering was performed using RaceId/StemId2 (Grün, D., L. Kester, and A. van Oudenaarden. 2014. “Validation of noise models for single-cell transcriptomics.” Nat. Methods 11 (6): 637–40.). Cells with less than 500 detected genes, less than 5000 aligned reads or more than 700 000 aligned reads were discarded. Only genes with a count of 5 in at least a single cell were used for analysis.
Genome_build: Gencode M11 / GRCm38
Supplementary_files_format_and_content: comma separated (csv) file with gene counts per sample
Submission date Oct 02, 2018
Last update date Feb 13, 2019
Contact name Ori Staszewski
Organization name University Medical Center Freiburg
Department Institute of Neuropathology
Street address Breisacher Str. 64
City Freiburg
ZIP/Postal code D-79106
Country Germany
Platform ID GPL13112
Series (2)
GSE120745 Single cell RNA-seq data of microglia cells from normal mouse brain at three ages (embryonic, 3 weeks and 16 weeks)
GSE120747 Single cell RNAseq data of CD1 microglia
BioSample SAMN10161727
SRA SRX4783818

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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