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GEO help: Mouse over screen elements for information. |
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Status |
Public on Dec 06, 2023 |
Title |
RNASeq, 2 days post-SCI-IP, biological sample 1 |
Sample type |
SRA |
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Source name |
OL translatome
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Organism |
Mus musculus |
Characteristics |
genotype: plp-cre;rpl22HA tissue: Mature oligodendrocyte (OL)
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Treatment protocol |
Spinal cord injury at the T9 level, using the IH impactor (50 kdyn) in 10-12 week old female mice
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Extracted molecule |
polyA RNA |
Extraction protocol |
Frozen tissue samples were homogenized with pre-chilled Dounce homogenizer (40 strokes using type A pestle, 40 strokes using type B pestle) in cold homogenization buffer on ice [RNAse-free water, 50 mM Tris (Sigma), pH 7.5, 100 mM KCl (Sigma), 12 mM MgCl2 (Sigma), 1% Nonidet P-40 (Sigma), 200 U/mL RNAsin (Promega, catalog #N2115), 1 mM DTT (Sigma-Aldrich), proteinase inhibitors (Roche), 1 mg/mL heparin (Sigma-Aldrich), 0.1 mg/mL cyclohexamide (Sigma-Aldrich)] to a wt/vol ratio of ~2-5%. Total RNA samples were also pooled respective to the OL samples for consistency. Homogenates were centrifuged at 10,000 x g for 10 minutes at 4°C to remove excess tissue. 10% of the cleared lysate was taken as input (IN, total spinal cord RNA) and stored at -80°C for subsequent analysis. The remaining lysate was incubated with monoclonal mouse anti-hemagglutinin (HA) antibody (HA.11 Clone 1612) for 4-6 h at 4°C in a microtube rotator and end-over-end gentle mixing. Protein A/G magnetic beads (Pierce) were prewashed in homogenization buffer and added to the lysates and further incubated at 4°C overnight with end-over-end mixing. The following day, samples were placed in a magnetic microtube stand on ice to isolate the magnetic beads, and the supernatant was stored in -80°C. Magnetic beads with the bound immunoprecipitated mRNA (IP) were washed 3 times using ice-cold high salt buffer solution (50 mM Tris, pH 7.5, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 100 g/mL cycloheximide) for at least 5 mins at 4°C and end-to-end mixing. Input and immunoprecipitated mRNA were purified using RNeasy Mini, RNA isolation kit (Qiagen, catalog # 74104) and Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, catalog # LSKIT0204) according to the manufacturers’ protocol, respectively. Concentration and quality of isolated RNA was first determined using a NanoDrop 1,000 spectrophotometer (Thermo Scientific). Then, the concentration was measured by Qubit Fluorometer (Thermo Scientific), and RNA integrity was assessed by capillary electrophoresis (Bioanalyzer, Agilent Technologies). Libraries were prepared using the Universal Plus mRNA-Seq (NuGEN Cat# 0508).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
2DPI-IP_S8
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Data processing |
Illumina BaseSpace FastQ v1.0.0 used for basecalling Raw sequences were checked for quality values using fastQC v0.10.1. Quality trimming was not deemed necessary. The sequences were directly aligned to the Mus musculus reference genome assembly (mm10) using Star version 2.6. Read counts for gene regions were obtained with HTSeq v.0.10.0 using Ensembl annotations (GRCm38.93). The annotation file was parsed to exclude mitochondria and ribosomal protein genes in an effort to reduce non-relevant variation in subsequent steps of the analysis. The resulting annotation file tested 21,965 gene locations. Assembly: GRCm38.p6
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Submission date |
Feb 14, 2023 |
Last update date |
Dec 06, 2023 |
Contact name |
Eric Christian Rouchka |
E-mail(s) |
eric.rouchka@louisville.edu
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Organization name |
University of Louisville
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Department |
Biochemistry and Molecular Genetics
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Lab |
KY INBRE Bioinformatics Core
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Street address |
522 East Gray Street
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City |
Louisville |
State/province |
Kentucky |
ZIP/Postal code |
40292 |
Country |
USA |
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Platform ID |
GPL19057 |
Series (1) |
GSE225308 |
Mature oligodendrocyte (OL) translatome changes at various times after moderate contusive spinal cord injury at the thoracic T9 level |
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Relations |
BioSample |
SAMN33293833 |
SRA |
SRX19367146 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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