|
| Status |
Public on Apr 10, 2020 |
| Title |
14_CC2_2_P7_m |
| Sample type |
SRA |
| |
|
| Source name |
Brain microglia
|
| Organism |
Mus musculus |
| Characteristics |
region: corpus_callosum
age: P07
gender: male
|
| Treatment protocol |
No treatment prior to cell isolation was performed.
|
| Growth protocol |
All animals were maintained in SPF condition
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Animals were killed by transcardial perfusion in anasthesia. Microglia cells were isolated from the brain by FACS sorting. Total RNA was extracted from sorted microglia by Pico Pure RNA extraction kit (Life Technologies) according to manufacturer’s protocol.
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).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
| |
|
| Description |
counts.txt
|
| 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 M10) using the Star aligner (version STAR_2.5.2b)
Gene counts were generated using featureCounts from the Subread package (featureCounts v1.6.2)
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 3.38).
Genome_build: GRCm38
Supplementary_files_format_and_content: Tab delimited text files with raw transcript counts ("counts.txt")
|
| |
|
| Submission date |
Jun 13, 2019 |
| Last update date |
Apr 10, 2020 |
| Contact name |
Ori Staszewski |
| E-mail(s) |
ori.staszewski@uniklinik-freiburg.de
|
| 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 |
GPL15103 |
| Series (1) |
| GSE132688 |
Unique microglia expression profile in developing white matter. |
|
| Relations |
| BioSample |
SAMN12052325 |
| SRA |
SRX6066743 |
