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Status |
Public on Oct 02, 2024 |
Title |
postnatal day 21 annulus fibrosus tissue sample 1 |
Sample type |
SRA |
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Source name |
annulus fibrosus
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Organism |
Mus musculus |
Characteristics |
tissue: annulus fibrosus strain: C57BL/6 genotype: wild-type age: postnatal day 21
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Extracted molecule |
total RNA |
Extraction protocol |
Care was taken to isolate AF tissue and discard NP and surrounding CEP. Each sample was flash-frozen in liquid nitrogen followed by homogenization using a mikro-dismembrator (Sartorius; Göttingen, Germany). RNA extracted from homogenized AF tissue through the TRIzol/chloroform followed by RNeasy column kit (Qiagen). RNA quality and concentration analyzed using Nanodrop 2000 (ThermoFisher Scientific). DNAse treatment performed on all samples. libraries were prepared using rRNA depletion method The sequencing libraries were clustered on a lane of a NovaSeq 6000 S4 flowcell. After clustering, the flowcell was loaded on the Illumina instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Control software. Raw sequence data (.bcl files) generated the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence The sequencing libraries were clustered on a lane of a NovaSeq 6000 S4 flowcell. After clustering, the flowcell was loaded on the Illumina instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the Control software. Raw sequence data (.bcl files) generated the sequencer were converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
The sequencing libraries were multiplexed and clustered on 4 flowcell lanes. After clustering, the flowcell was loaded on the Illumina HiSeq (4000 or equivalent) instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification. After demultiplexing, sequence data was checked for overall quality and yield. Then, raw sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The reads were then mapped to the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the groups of samples was performed. The Wald test was used to generate P values and Log2 fold changes. Genes with adjusted P values < 0.05 and absolute log2 fold changes >1 were called as differentially expressed genes for each comparison. Gene ontology analysis was performed on the statistically significant set of genes by implementing the software GeneSCF. The GO list was used to cluster the set of genes based on their biological process and determine their statistical significance. assembly: GRCm38 processed data files format and content: normalized_counts.csv
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Submission date |
Aug 25, 2024 |
Last update date |
Oct 02, 2024 |
Contact name |
Danielle D'Erminio |
E-mail(s) |
derminiodanielle@gmail.com
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Phone |
5135081064
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Organization name |
Mount Sinai
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Department |
Orthopaedics
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Lab |
Iatridis
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Street address |
1 Gustave L. Levy Place
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City |
Manhattan |
State/province |
New York |
ZIP/Postal code |
10029 |
Country |
USA |
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Platform ID |
GPL13112 |
Series (1) |
GSE275620 |
Comparison of annulus fibrosus tissue from tails of neonatal mice aged postnatal day 5, day 14, day 21, and day 28 |
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Relations |
BioSample |
SAMN43357651 |
SRA |
SRX25820609 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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