|
| Status |
Public on Nov 12, 2020 |
| Title |
Knock_down2 |
| Sample type |
SRA |
| |
|
| Source name |
Cerebral cortex
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
gender: Male
age: 9-12 weeks
treatment: eRNA_06347 knockdown
|
| Treatment protocol |
In order to understand the role of eRNAs in mediating the post-stroke pathophysiology in vivo, we knocked-down the expression of eRNA_06347 in the mouse brain using sequence-specific locked nucleic acid (LNA) antisense oligos delivered via the lateral ventricles. Cohorts of mice underwent either eRNA_06347 knockdown or Negative control oligo treatment, followed by a 1 h Middle cerebral arterey occlusion(MCAO) and 6 h of reperfusion (n=3/group). A sham cohort (n=3/group) receiving undergoing sham surgery served as healthy controls.Transient focal ischemia was induced using the MCAO method. Mice were anesthetized with 5% isoflurane and a midline neck incision was made to expose the common carotid artery (CCA). A nick was made in CCA and a 6-0 silicon-coated filament was inserted and advanced through the internal carotid artery up to the middle cerebral artery (MCA) and left in place for 1 h to occlude blood flow into the MCA. Cerebral blood flow and body tem- perature were continuously monitored. The incision was sutured and the animals were returned to warm cages for recovery. After 1 h of occlusion, the filament was removed, the incision was sutured, and the mice were returned to their cages. Reperfusion was allowed for 6 h. For the sham cohort, midline neck incision was similarly done to expose the ECA; however, no filament was inserted to occlude the MCA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Animals were euthanized and the infarcted ipsilateral cortical tissues were immediately harvested and snap-frozen in liquid nitrogen before storing in -80°C. Total RNA was then isolated from the tissues using the mirVana total RNA isolation kit, followed by Dnase I tratment. The RNA integrity was evaluated using Bioanalyzer.
For Negative control and Knockdown samples Poly(A) RNA sequencing, the library was prepared following Illumina’s truseq-stranded-mRNA sample preparation protocols. RNA integrity of total RNA sample received was checked using an Agilent Technologies 2100 Bioanalyzer. The polyA containing mRNA molecules were purified using poly-T oligo attached magnetic beads and using two rounds of purification. After purification the polyA RNA was fragmented using divalent cation containing buffer and elevated temperature. Quality control analysis and quantification of the DNA library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-end sequencing was performed on Illumina’s NovaSeq 6000 sequencing system.For the Sham samples,previously generated data from our lab was used. Please refer to GEO accession number GSE112348 for the relevant details regarding sham samples.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
The sequencing reads were aligned to the mouse reference genome GRCm38 using HISAT2 (v2.0). StringTie (v1.3) was then used to assemble the transcripts of each sample independently using mouse transcript annotations from Ensembl 81 as a guide.StringTie and gffcompare was then used to merge all the assemblies to create a StringTie merged gtf. Finally, the mRNA profiling was done using StringTie and Differentially expressed mRNAs were identified using Rpackage Ballgown.
Genome_build: mm10
Supplementary_files_format_and_content: gtf files
|
| |
|
| Submission date |
Aug 27, 2019 |
| Last update date |
Nov 12, 2020 |
| Contact name |
Ashutosh Dharap |
| E-mail(s) |
ashutosh.dharap@hackensackmeridian.org
|
| Organization name |
HackensackMeridian Health JFK Medical Center
|
| Department |
Neuroscience
|
| Lab |
Laboratory for Stroke Research and Noncoding RNA Biology
|
| Street address |
65 James st
|
| City |
Edison |
| State/province |
NJ |
| ZIP/Postal code |
08820 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE136405 |
Gene expression changes following Knock-down of eRNA_06347 in post-stroke mouse cortex |
|
| Relations |
| BioSample |
SAMN12642308 |
| SRA |
SRX6774370 |
