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| Status |
Public on Feb 13, 2019 |
| Title |
P212_L5 |
| Sample type |
SRA |
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| Source name |
WT_forebrain_embryonal
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| Organism |
Mus musculus |
| Characteristics |
tissue: forebrain
age: embryonal
replicate_id: WT_forebrain_embryonal_2
strain: CD1
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| 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)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
geosubmission_counts.csv
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| 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
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| Submission date |
Oct 02, 2018 |
| Last update date |
Feb 13, 2019 |
| Contact name |
Ori Staszewski |
| E-mail(s) |
ori.staszewski@uniklinik-freiburg.de
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| Organization name |
University Medical Center Freiburg
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| Department |
Institute of Neuropathology
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| Street address |
Breisacher Str. 64
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| City |
Freiburg |
| ZIP/Postal code |
D-79106 |
| Country |
Germany |
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| 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 |
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| Relations |
| BioSample |
SAMN10161793 |
| SRA |
SRX4784085 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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