 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 16, 2021 |
| Title |
E5386 - ALS Cre Control |
| Sample type |
SRA |
| |
|
| Source name |
Lumbar Spinal Cord
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Lumbar Spinal Cord
cell type: Bulk
strain: C57BL/6
age: 120 days
genotype: SOD1G93A Ager+/+ Cx3cr1Cre/+
|
| Treatment protocol |
Tamoxifen was used to delete Ager at 3 months of age. All animals were administered 0.2 μg of tamoxifen (TAM, Sigma, Cat: T5648), once daily every day for a total of five intraperitoneal injections at 3 months (12 weeks) of age. These mice were sacrificed at the age of 4 months. All animals were then deeply anaesthetized using Ketamine and Xylazine and humanely sacrificed and utilized for post-mortem molecular analyses.
|
| Growth protocol |
All experiments were performed on male C57BL/6J mice intercrossed with the following strains: B6.129P2(Cg)-CX3CR1tm2.1(cre/ERT2)Litt/WganJ (JAX Stock No: 021160), Ager floxed mice in wildtype or B6.Cg-Tg(SOD1G93A)1Gur/J (JAX Stock No:004435) backgrounds. All mice were maintained under pathogen-free conditions and all experiments were performed under protocols approved by the New York University School of Medicine Institute Animal Care Committee (IACUC) in accordance with international and NIH guidelines. All mice were housed in a temperature- (19–23 °C) and humidity-regulated (30–70% relative humidity) environment with a 12/12 h light/dark cycle and received standard chow food (Lab Diets, Cat: 5053) pellets and water ad libitum.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were anesthetized with Ketamine and Xylazine. The spinal column was rapidly removed, and the lumbar spinal cord was retrieved. Lumbar spinal cords were flash frozen and stored at -80˚C until processed. Frozen tissue was kept on dry ice until homogenized in Qiazol reagent (Qiagen Inc, Cat: 79306). Crude homogenate was mixed with chloroform (70µL per 350µL homogenate) to facilitate removal of lipids, and allowed to equilibrate at room temperature for 2 min. Spun at 13,000 x g for 15 min at 4˚C. The aqueous phase containing the RNA was transferred to a new tube. Then the following steps were completed as detailed by the manufacturer’s instructions using an RNeasy Mini Kit (Qiagen, Cat: 74104) with on-column DNAse digestion (Qiagen, Cat:79254). RNA concentration was determined via a NanoDrop spectrophotometer (ThermoFisher, Model: ND-1000) and RNA integrity (RIN) was measured using RNA 6000 Pico Kit in a 2100 Bioanalyzer (Agilent).
High quality lumbar spinal cord RNA samples, as confirmed by Bioanalyzer, (RINs: 7.9-8.7) free of DNase and RNase were prepared for sequencing using the TruSeqStranded mRNA library prep kit (Illumina, Cat: 20020594) and the NovaSeq 6000 SP Reagent Kit v1.5 (Illumina, Cat: 20028401). High-throughput RNA sequencing (RNA-seq) was completed using an Illumina NovaSeq 6000 sequencer performed by the NYU Grossman School of Medicine Genome Technology Core.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Trimmed low quality bases and removed adapter contamination with Trimmomatic 0.36 with parameters ILLUMINACLIP:adapters.fa:2:30:10:1:true TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:35
Aligned reads to the mouse mm10 genome build using STAR 2.7.3a including parameters --outFilterMismatchNoverLmax 0.2 --outFilterMultimapNmax 1 --outFilterType BySJout --outSAMstrandField intronMotif --outSAMattributes NH HI AS nM NM MD XS --outSAMmapqUnique 60 --twopassMode Basic --quantMode GeneCounts --outSAMtype BAM Unsorted --outStd BAM_Unsorted
Sorted BAM files with samtools sort 1.9
Counted reads per gene using featureCounts function in subread 1.6.3 with parameter -p for paired-end
Normalized read counts by library size as log2(counts per million) using edgeR 3.26.8
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files with raw read pair counts and log2(CPM) normalized read pair counts per gene (rows) per sample (columns)
|
| |
|
| Submission date |
Oct 29, 2020 |
| Last update date |
Jun 16, 2021 |
| Contact name |
Paul Francis Gugger |
| E-mail(s) |
[email protected]
|
| Organization name |
NYU Langone Health
|
| Department |
Medicine
|
| Street address |
435 E 30th St
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE160402 |
Microglia RAGE exacerbates the progression of neurodegeneration within the SOD1G93A murine model of amyotrophic lateral sclerosis in a sex-dependent manner |
|
| Relations |
| BioSample |
SAMN16595279 |
| SRA |
SRX9398723 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
