|
| Status |
Public on Nov 18, 2024 |
| Title |
MCAO_24h_B |
| Sample type |
SRA |
| |
|
| Source name |
brain
|
| Organism |
Mus musculus |
| Characteristics |
tissue: brain
condition: MCAO
timepoint: 24 hours
replicate: B
|
| Treatment protocol |
Prior to the induction of MCAO, mice were anaesthetized with 3% isoflurane (Aerrane, Baxter) and amaintained in 2% isoflurane using a vaporizer (Tec-3, Cyprane Ltd.). A skin incision between the orbit and the external auditory meatus was made, and a 1-2 mm hole was drilled through the frontal bone 1 mm rostral to the fusion of the zygoma and the squamosal bone, about 3.5 mm ventrally to the dorsal surface of the brain. The middle cerebral artery (MCA) was exposed after the dura was opened and removed. The MCA was occluded by short coagulation with bipolar tweezers (SMT) at a proximal location, followed by transection of the vessel to ensure permanent occlusion. During the surgery, body temperature was maintained at 37 ± 1C using a heating pad. This MCAO model yields small infarct lesions in the parietal cortical region. Intact cortical tissue from 3 months old mice was used as control.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Samples were collected from the injured cortical region (including affected as well as non-affected brain tissue) and homogenized using the TissueLyser (QIAGEN). Total RNA was extracted with TRI Reagent (Sigma-Aldrich) according to the manufacturer’s protocol and treated with TURBO DNA-free kit (Thermo Fisher). RNA quantity and purity was assessed using the NanoDrop 2000 spectrophotometer (Thermo Fisher) and RNA integrity was assessed using the Fragment Analyzer (Agilent). All samples had RQN > 8.
Libraries were prepared from 400 ng total RNA with QuantSeq 30 Library Prep Kit FWD (Lexogen) according to manufacturer’s protocol. 1 µl of ERCC spike-in (c = 0.01x; Thermo Fisher) per library was included. Libraries were quantified on the Qubit 2 fluorometer (Thermo Fisher) and Fragment Analyzer (Agilent), and sequenced on the NextSeq 500 high-output (Illumina) with 85 bp single-end reads. 11.5 – 38 million reads were obtained per library with a median of 16 million reads
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
MCAO_24h_B
|
| Data processing |
Adaptor sequences and low-quality reads were removed using TrimmomaticSE v0.36. Reads mapping to mtDNA and rRNA were filtered out using SortMeRNA v2.1 with default parameters. The remaining reads were aligned to GRCm38 and ERCC reference using STAR v2.5.2b with default parameters81. Mapped reads were counted over Gencode vM8 gene annotation using htseq-count with union mode for handling of overlapping reads.
We filtered the data for genes with >10 reads and normalized them by varianceStabilizingTransformation function. We applied the DESeq model using DESeq function with type of fit parameter set to ‘local’.
Assembly: mm10/GRCm38
Supplementary files format and content: tab-delimited text file containing DESeq normalized expression matrix.
|
| |
|
| Submission date |
May 31, 2023 |
| Last update date |
Nov 18, 2024 |
| Contact name |
Lukas Valihrach |
| Organization name |
Institute of Biotechnology, Czech Academy of Science
|
| Lab |
Laboratory of Gene Expression
|
| Street address |
Prumyslova 595
|
| City |
Vestec |
| ZIP/Postal code |
25250 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE233811 |
Spatiotemporal transcriptomic map of glial cell response in a mouse model of acute brain ischemia [bulk RNA-Seq] |
| GSE233815 |
Spatiotemporal transcriptomic map of glial cell response in a mouse model of acute brain ischemia |
|
| Relations |
| BioSample |
SAMN35546758 |
| SRA |
SRX20554172 |
