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| Status |
Public on Jan 31, 2021 |
| Title |
01W_C_05 |
| Sample type |
SRA |
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| Source name |
sham surgery_1wk_gray matter of spinal cord
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: Long Evans Crl:LE
age: adult (>10 weeks)
surgery: sham surgery
time point of tissue dissection in weeks: 1
tissue: gray matter of spinal cord
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| Treatment protocol |
At 1, 3, 6 or 12 weeks after surgery, animals were deeply anaesthetized with 5% isoflurane followed by an intraperitoneal injection of Esconarkon (600 mg/kg body weight, Streuli Pharma AG, Switzerland) and transcardially perfused with ice cold Ringer solution (Braun, Germany) supplemented with Heparin (100 000 IU/l, Roche Pharmaceuticals, Switzerland), 0.017% NaHCO3 and 0.25% NaNO2. Immediately afterwards, a 1 mm thick transverse slice of the spinal cord at vertebral level C3 was prepared, and the bilateral intermediate laminae (laminae IV-VII) were dissected. The tissue was immediately frozen in liquid nitrogen. Samples were stored in -80°C until RNA extraction. For the histological assessment of the lesion site, spinal cord tissue containing the lesion site was dissected and incubated in 4% paraformaldehyde (PFA) supplemented with 5% sucrose for 24 hours. Tissue was then transferred into a 0.1M phosphate-buffered solution containing 30% sucrose for cryoprotection and stored until histological processing.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the Nucleospin RNA XS kit (Macherey-Nagel, Germany). Briefly, tissue samples were lysed in 1 ml TRIzol reagent (Thermo Fisher Scientific, USA) and mechanically disrupted using a homogenizer. Chloroform was added and the aqueous phase containing the RNA was added onto the Nucleospin column. From there the manufacturer’s protocol was followed including the DNAse treatment. The quality and quantity of the isolated total RNA was determined with a Qubit 2.0 Fluorometer (Life Technologies, USA) and a Tapestation 2200 (Agilent, Germany)
cDNA libraries were prepared by the Functional Genomics Center Zürich (University and ETH Zürich) according to the procedure described as follows: TruSeq Stranded Total RNA Library Prep Kit (Illumina Inc, USA) was used in the following steps. Briefly, the 200 ng total RNA samples were ribo-depleted using Ribo Zero Gold (Illumina Inc, USA) and fragmented with divalent cations and an elevated temperature. The fragmented RNA was then reverse-transcribed into cDNA with Actinomycin, which prevents DNA-dependent synthesis but allows RNA-dependent synthesis. Actinomycin is added during first strand synthesis. To achieve strand specificity, dUTP was incorporated instead of dTTP in the course of the second strand synthesis. The polymerase used for the subsequent PCR amplification will not incorporate past dUTP and so the second strand will be suppressed. A single ‘A’ nucleotide is added to the 3’ ends of the cDNA fragments before ligation of the TruSeq adapters. The adapters contain the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using qPCR (LightCycler 96, Roche, Switzerland) and Tapestation 2200 (Agilent, Germany). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. Cluster Generation and sequencing were performed by the Functional Genomics Center Zürich (University and ETH Zürich) according to the procedure described as follows: The TruSeq SR Cluster Kit v4-cBot-HS (Illumina Inc, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 4000 single end 125 bp using the TruSeq SBS Kit v4-HS (Illumina Inc, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
20170831.A-01W_C_05
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| Data processing |
Fastq files were generated with the illumina software bcl2fastq v2.19.05. All subsequent analyses were run through the Sushi web framework (Hatakeyama, M., Opitz, L., Russo, G., Qi, W., Schlapbach, R., & Rehrauer, H. (2016). SUSHI: An exquisite recipe for fully documented, reproducible and reusable NGS data analysis. BMC Bioinformatics, 17(1), 1–9.)
Reads were trimmed with trimmomatic (Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114–2120.) to remove 3'-adapters
Reads were aligned with the STAR aligner v2.5.3a with the options '--twopassMode None --runThreadN 8 --outFilterType BySJout --outFilterMatchNmin 30 --outFilterMismatchNmax 10 --outFilterMismatchNoverLmax 0.05 --alignSJDBoverhangMin 1 --alignSJoverhangMin 8 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outFilterMultimapNmax 50 --chimSegmentMin 15 --chimJunctionOverhangMin 15 --chimScoreMin 15 --chimScoreSeparation 10 --outSAMstrandField intronMotif --outSAMattributes'
Strand-specific (reversely stranded) read counting was performed using featureCount (subread, version 1.2.6) (Liao, Y., Smyth, G. K., & Shi, W. (2013). The Subread aligner: Fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Research, 41(10)).
Genome_build: Ensembl/Rnor_6.0
Supplementary_files_format_and_content: text files holding read counts per gene
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| Submission date |
Jan 30, 2018 |
| Last update date |
Feb 01, 2021 |
| Contact name |
Natalie Russi |
| Organization name |
University of Zurich and ETH Zurich
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| Department |
Brain Research Institute
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| Lab |
Research group of Prof. Dr. Martin E. Schwab
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| Street address |
Winterthurerstrasse 190
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| City |
Zuerich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL22396 |
| Series (1) |
| GSE109902 |
Transcriptional screen in the target region of sprouting hindlimb corticospinal fibers after thoracic spinal cord injury in rats |
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| Relations |
| BioSample |
SAMN08442107 |
| SRA |
SRX3633146 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2973531_01W_C_05.txt.gz |
149.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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