|
| Status |
Public on Jul 15, 2015 |
| Title |
wt_micro_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Wild-type microglia
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Wild-type microglia
genotype: Wild-type microglia
strain: DTR
|
| Treatment protocol |
Tamoxifen (TAM; Sigma) was suspended on 37°C in olive oil for 60 mins (MP Biomedicals). 2 mg Tamoxifen was administered subcutaneously (s.c.) twice on postnatal days 12 and 14. For microglia depletion, mice were injected intraperitoneally (i.p.) with 500ng diphtheria toxin (DT; Merck Millipore) three times, with a one-day interval between each injection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were anesthetized and perfused with NaCl and organs were excised out. Brains were either fixed with 4% of PFA for histology, snap frozen in liquid nitrogen for RNA preparation, or digested according to manufacturer’s guidelines supplied in the MACS tissue dissociation kit Papain (Miltenyi Biotec). The tissue homogenate was loaded on a 30:70% Percoll gradient for enrichment of CNS infiltrates. The interphasic cellular ring was collected for further investigation. The different microglia samples were FACS sorted (FACS Aria) and immediately centrifuged down for 15 mins and 400 x g. RNA was isolated with the RNeasy® Plus Micro Kit (Qiagen) according to the manufacturer’s instructions. RNA amounts were measured by Q-bit 2.0 fluorometer (Life Technologies) and the quality was assessed on a Bioanalyzer (Agilent) using a High Sensitivity Chip. Samples with a RNA integrity number (RIN) > 8 were used for library preparation with TruSeq® RNA Sample Prep v2 Kit (Illumina) according to the manufacturer’s manual. Library size distribution was assessed on a Bioanalyzer 2100 using a High Sensitivity DNA Chip (Agilent).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Sequenced reads were mapped to the mouse genome reference sequence (mm10, retrieved from Illumina iGenomes); TopHat version 2.0.10, combined with Bowtie2 (2.1.0.0) was used for this purpose. Default parameters were used to perform the alignment.
Quantification of gene expression was performed with the htseq-count tool from the HTSeq software suite. The corresponding annotation was also retrieved from the Illumina iGenomes archive.
Differential expression analysis was performed by using DESeq (version 1.14.0). A False Discovery Rate (FDR) cutoff of 0.05 was selected.
Genome_build: mm10
Supplementary_files_format_and_content: Tag-delimited text file, containing normalized read counts, stored in a NxM matrix (N genes, M samples) with a header line
|
| |
|
| Submission date |
Apr 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ari Waisman |
| E-mail(s) |
waisman@uni-mainz.de
|
| Organization name |
Institute for Molecular Medicine
|
| Street address |
Obere Zahlbacher Straße 67
|
| City |
Mainz |
| ZIP/Postal code |
55131 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE68376 |
Interleukin-1 signaling is involved in local self-renewal and maintenance of microglia |
|
| Relations |
| BioSample |
SAMN03571208 |
| SRA |
SRX1013025 |
