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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 13, 2024 |
| Title |
9L1, d6 |
| Sample type |
SRA |
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| Source name |
bone (femoral head)
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| Organism |
Homo sapiens |
| Characteristics |
individual: HUK9
disease state: osteoporosis
tissue: bone (femoral head)
cell type: human mesenchymal stem cells (hMSC)
treatment: Transfected with L1 RNA
time: d6
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| Treatment protocol |
Synthetic L1 RNA was transfected in MSC differentiating to osteoblasts at day 5 using LipofectamineTM MessengerMAXTM (Invitrogen, USA, Cat. No. LMRNA003) with a modified protocol for low RNA amount. RFP mRNA (System Bioscience, USA, Cat. No. MR800A-1) was used as negative control
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| Growth protocol |
Human MSC isolated from femoral head were incubated in DMEM (PAN Biotech, Germany, Cat. No. P04-03550) supplemented with 1% (v/v) 100× Penicillin-Streptomycin Solution (Biowest, France, Cat. No. L0022), 10% (v/v) FCS (Biowest, France, Cat. No. S181S), 1% (v/v) 200mM L-Glutamine solution (Sigma, USA, Cat. No. G7513), 5ng/ml FGF-2 (Sigma, USA, Cat. No. F0291) and 10ng/ml FGF-4 (Sigma, USA, Cat. No. F8424) in a humidified atmosphere containing 5% CO2. To induce osteogenic differentiation, MSC were seeded on Nunc™ 24-well plates (Thermo Fisher, USA, Cat. No. 142475) or 48-well plates (Thermo Fisher, USA, Cat. No. 150687) at a density of 1.5 × 104 cells/cm2. After 24 h, differentiation was induced using StemPro® Osteogenesis Kit (Gibco/Life Technologies, USA, Cat. No. A10072-01). The medium was exchanged every 3 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested and resuspended in 1ml of QIAzol Lysis reagent (Qiagen, Cat. No.79306). Total RNA was then purified with RNeasy Plus Mini kit (Qiagen, cat. No. 74134) with minimal modifications to manufacturer’s instructions. DNase treatment (RNase free DNase set, Qiagen, Cat. No. 79254) was performed to remove any residual DNA. RNA quality and concentration were checked using a NanodropTM 2000 spectrophotometer (ThermoFisher).
Total RNA-seq library was prepared with CORALL Total RNA library prep with RiboCop rRNA for Human/Mouse/Rat depletion kit (Lexogen GmbH, Vienna, Austria) following manufacturer’s instructions (library type: fr-secondstrand) by IGA Technology service (Italy). Final libraries were checked with both Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent Bioanalyzer DNA assay or Caliper (PerkinElmer, Waltham, MA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
61692_ID1885_5-9L1d6-A001-E01_S75_L004_R1_001
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| Data processing |
RNA-seq read quality control (QC) analyses and filtering of high-quality reads were executed using FastQC and BBDuk (Bushnell et al. 2017) by setting a minimum read length of 35 bp and a minimum Phred-quality score of 25. After trimming quality control, high quality reads were aligned to the mouse genome reference (GrCm38p6/mm10) with STAR 2.7.3a (Dobin et al. 2012), while FeatureCounts 1.6.3 package (Liao et al. 2014) was used to assign reads to genes.
Genes with low, constant levels of raw counts across one or more experimental conditions were filtered to eliminate the “uninformative” genes using HTSFilter 1.30.1 (Rau et al. 2013). Filtered gene data was further processed with the EdgeR package (Robinson et al. 2009) to normalize the raw counts.
Assembly: GRCh38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample
Supplementary files format and content: Matrix table with fpkm normalized gene counts for every gene and every sample
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| Submission date |
Apr 28, 2022 |
| Last update date |
Sep 13, 2024 |
| Contact name |
Alfonso Saera-Vila |
| Organization name |
Sequientia Biotech
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| Street address |
Dr. Trueta 179
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| City |
Barcelona |
| ZIP/Postal code |
08005 |
| Country |
Spain |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE201774 |
LINE-1 retrotransposons are reduced in bone of osteoporotic patients, correlate positively to BMD, and stimulate bone anabolism in transfected mesenchymal stem cells |
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| Relations |
| BioSample |
SAMN27926701 |
| SRA |
SRX15038770 |
| 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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