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| Status |
Public on Nov 07, 2017 |
| Title |
Lrp5/6KO+Wnt3a rep2 |
| Sample type |
SRA |
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| Source name |
Osteoblasts
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| Organism |
Mus musculus |
| Characteristics |
genotype: Lrp5fl/fl;Lrp6fl/fl;UBC-Cre-ERT2
age: 5-6 days
tissue: Calverial osteoblasts
treatment: hydroxytamoxifen (TMX), Wnt3a
experimental batch: 5
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| Treatment protocol |
Osteoblasts (OBs) were isolated from calvaria of neonates (5-6 days old) by serial digestion in Collagenase 1 and EDTA solutions. These cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C. Following 24h incubation the media was changed to remove digestion debris and non-viable cells and these cultures were treated with 100 ng/ml recombinant Wnt3a (R&D systems, Minneapolis, MN, USA). The RNA was isolated from these cultures after incubating the cells for another 24h. To generate Lrp6 and dual Lrp5/6 deficient OBs, mice carrying Lrp5 (Lrp5fl/fl) and Lrp6 (Lrp6fl/fl) conditional alleles were crossed to a tamoxifen inducible-ubiquitous Cre recombinase transgenic strain of mice (UBC-Cre-ERT2; Jackson Laboratories, Bar Harbor, ME, USA) to generate Lrp6fl/fl;UBC-Cre-ERT2 and Lrp5fl/fl;Lrp6fl/fl;UBC-Cre-ERT2 mice . Following isolation of osteoblasts from Lrp6fl/fl; UBC-Cre-ERT2 and Lrp5fl/fl;Lrp6fl/fl;UBC-Cre-ERT2, cells were cultured for 24h in DMEM+FBS+P/S media, followed by 48h treatment with DMEM+FBS+P/S with 1 uM hydroxytamoxifen (Sigma-Aldrich, St. Louis, MO, USA) prior to 24h Wnt3a treatment.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using RNeasy Mini Kit (QIAGEN Inc, Germantown, MD, USA) according to manufacturer’s protocol.
Poly(A)+-enriched cDNA libraries were generated using the Illumina TruSeq Sample Preparation kit (Illumina Inc, Hayward, CA, USA) according to manufacturer’s protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
Sequenced reads were trimmed for low-quality sequence, then mapped to mouse genome mm10 using TopHat (version 2.0.11)
After read mapping, ‘featureCounts’ from Rsubread package (version 1.22.2) was used to generate gene-wise read counts.
Genes were filtered from downstream analysis if they did not have a CPM (counts per million mapped fragments) value of at least 2 in at least five libraries
Data was normalized using edgeR and experimental batch effects were removed using 'removeBatchEffect' function in edgeR
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include log2 CPM values for each Sample .
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| Submission date |
Sep 05, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Aimy Sebastian |
| E-mail(s) |
[email protected]
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| Organization name |
Lawrence Livermore National Laboratory
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| Street address |
7000 East Ave
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| City |
Livermore |
| ZIP/Postal code |
94550 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (1) |
| GSE103492 |
Transcriptome analysis of Wnt3a-treated Wild Type, Lrp5KO , Lrp6KO and Lrp5/6KO osteoblasts |
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| Relations |
| BioSample |
SAMN07605558 |
| SRA |
SRX3161487 |
| 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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