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| Status |
Public on Feb 25, 2021 |
| Title |
BOTH1: BM_RNASeq - Bone_Marrow Combined_treatment replicate_1 |
| Sample type |
SRA |
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| Source name |
Bone marrow
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 6-7 weeks
tissue: Bone marrow
treatment: ZA & G-CSF
biological replicate: 1
sequencing batch: NA
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| Treatment protocol |
Zoledronic acid (ZA) [1-hydroxy-2-(1H-imidazole-1-yl)ethylidene-bisphosphonic acid] (Novartis Pharmaceuticals, Cambridge, MA) was dissolved in 1X Hank’s Balanced Buffer Solution (Gibco) and stored at 4°C until use. ZA was administered to mice at a dose of 100 µg/kg (i.p.). Recombinant human granulocyte colony stimulating factor (G-CSF) (carrier-free, Biolegend #578604) was administered to nude mice each day for 3 days at a dose of 50 µg/kg (i.p.) beginning one day after administration of ZA, and C57BL/6 mice each day for 3 days at a dose of 50 µg/kg (i.p.) beginning 2 days after administration of ZA. For G-CSF depletion experiments, nude mice were treated with 100 µg/kg (i.p.) G-CSF antibody (R&D Systems, MAB414100) or the 100 µg/kg (i.p.) isotype control IgG (R&D Systems, MAB005) six hours prior to intracardiac injection of tumor cells.
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| Growth protocol |
6-7 week old C57BL/6J female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal procedures were performed in accordance with the ethics and regulations of Brigham & Women’s Hospital Institutional Animal Care and Use Committee (protocol approval 2017N000056), Boston Children’s Hospital Institutional Animal Care and Use Committee (protocol approval 12-11-2308R), and Massachusetts General Hospital (protocol approval 2017N000023).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolated from mouse bone marrow was subject to quality control using the Bioanalyzer (Agilent). BMCs were prepared for flow cytometry by suspension in sterile PBS containing 2% FBS. Cells were labeled with appropriate antibodies for 30 minutes at 4°C. Gating was used to exclude debris, cell clumps, and dead cells (using 7-aminoactinomycin D; 7-AAD Viability Staining Solution (BioLegend, 420404).Prior to sorting of the M/OCP populations, the Murine Direct Lineage Cell Depletion Kit (Miltenyi Biotec Inc., 130-110-470) was used to enrich for Lin- populations, which was confirmed by flow cytometry for the markers in the lineage cocktail. Myeloid/osteoclast progenitor cell (M/OCP) populations were defined as Lineage- CD115+. Antibody panel includes: Pacific BlueTM anti-mouse Lineage Cocktail (BioLegend, 133310: CD3-, Ly-6G/Ly6-C-, Cd11b-, CD45R-, TER119-) and Alexa Fluor® 488 anti-mouse CD115 (CSF-1R; BioLegend, 135511).
RNA was converted into a DNA library using the Ultra RNA Library Prep Kit for Illumina (New England BioLabs). After further quality control, libraries were sequenced on the HiSeq 2000 (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sample used in bone marrow RNAseq comparison
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| Data processing |
Pseudoalignment with salmon, version ,0.8.2
Import and summarize salmon generated counts with tximport, version 1.6.0
Batch correct for sequencing run with RUVSeq and replicate samples, k=3(for OCP-RNASeq analysis), retain replicate sample with most reads
Analyze differential expression with DESeq2 BioConductor package, version 1.16.1 (using batch correction factors from RUVSeq in the OCP-RNASeq analysis)
Genome_build: mm10
Supplementary_files_format_and_content: comma separated format, rawCounts matrix file contains raw counts as generated by Salmon and imported by tximport (these counts were used for the DESeq2 analysis), tpm matrix contains transcripts per million normalized counts as calculated by tximport
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| Submission date |
May 15, 2018 |
| Last update date |
Feb 25, 2021 |
| Contact name |
Jessalyn Ubellacker |
| E-mail(s) |
jubellacker@fas.harvard.edu
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| Organization name |
Harvard University
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| Lab |
Sandra McAllister Laboratory
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| Street address |
4 Blackfan Circle
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE108250 |
Modulating Bone Marrow Hematopoietic Lineage Potential to Prevent Breast Cancer Bone Metastasis |
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| Relations |
| BioSample |
SAMN09210202 |
| SRA |
SRX4084032 |
| 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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