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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 03, 2018 |
| Title |
Wild-type (WT) LPS stimulated [5365_MG] |
| Sample type |
SRA |
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| Source name |
FACS-sorted microglia, WT, LPS
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| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype/variation: WT
treatment: LPS
tissue: Brain
cell type: microglia
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| Treatment protocol |
Gene Knockout in microglia-specific A20 knockout mice, i. p. treatment with LPS (3.5 mg/kg).
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| Growth protocol |
CD11b+ CD45Int microglia were sorted in RNAprotect [from: RNeasy Plus Micro Kit (QIAGEN, Hilden, Germany)] cell reagent using FACS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were stored and shipped in RNAprotect buffer at 2-8 °C. After pelleting, the RNAprotect buffer was replaced by RLT Plus and the samples were homogenized by vortexing for 30 sec. Genomic DNA contamination was removed using gDNA Eliminator spin columns. Next, ethanol was added and the samples were applied to RNeasy MinElute spin columns followed by several wash steps. Finally, total RNA was eluted in 12 μl of nuclease-free water. Purity and integrity of the RNA were assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico LabChip reagent set (Agilent, Palo Alto, CA, USA).
The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from 1 ng total-RNA. Double-stranded cDNA was amplified by LD PCR (11 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step, 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
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| Description |
5365_MG
Processed data file: counts.txt
Processed data file: cpm.txt
Processed data file: Voom_Matrix.txt
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| Data processing |
Basecalling was performed using RTA 1.18.61.
bcl files were converted to fastq using CASAVA 1.8.2.
Fastq files were aligned to the mouse genome (Gencode M11) using the Star aligner (version STAR_2.5.2b).
Gene counts were generated using featureCounts from the Subread package (featureCounts v1.5.1).
Prior to differential gene expression analysis, low gene counts were removed with the lowest 32% of genecounts (sums of all samples) being removed.
Count normalization and differential gene expression analysis was performed using the limma/voom pipeline (limma version 3.28.21).
Genome_build: GRCm38
Supplementary_files_format_and_content: Tab-delimited text files with raw transcript counts ("counts.txt"), counts per million ("cpm.txt") and normalized counts ("Voom_matrix.txt").
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| Submission date |
Apr 07, 2017 |
| Last update date |
May 15, 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 |
GPL15103 |
| Series (2) |
| GSE97538 |
RNAseq data from FACS-sorted microglia from mouse brain from control mice (uninjected) or LPS-injected mice in the context of A20 knockout |
| GSE107733 |
Microglia-expressed A20 regulates synapse formation and inhibits inflammasome-dependent neuroinflammation |
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| Relations |
| BioSample |
SAMN06701611 |
| SRA |
SRX2731478 |
| 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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