C57BL/6 mice expressing a fusion eGFP-L10a ribosomal protein under control of the endothelial-specific promoter VE-cadherin (VEcadTRAP mice)16 (link) were crossed with ELTD1−/− mice. Mice were intracranially injected with GL261 cells, brains were harvested at day 23 after tumor injection, and tumors were dissected under a stereological microscope. Brains from healthy mice were used as controls. Endothelial ribosomes were purified and mRNA was extracted as described.18 (link),19 (link) Briefly, eGFP-tagged polysomes were isolated using anti-GFP-bound Dynabeads (Qiagen), endothelial cell mRNA was extracted using Trizol and purified by RNeasyMicro kit (Qiagen).
Dynabeads
Manufactured by Qiagen
Dynabeads are magnetic beads used in a variety of laboratory applications. They are designed to facilitate the isolation, separation, and purification of target molecules, such as proteins, nucleic acids, and cells, from complex biological samples. The core function of Dynabeads is to provide a versatile and efficient tool for researchers to extract and concentrate specific analytes of interest from complex mixtures.
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Lab products found in correlation
3 protocols using dynabeads
1
Endothelial Ribosome Profiling in Glioblastoma
2
Endothelial Translating Ribosome Capture
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The Translating Ribosomal Affinity Purification (TRAP) protocol was previously used for identification of cell-specific expression.32 In the TRAP mice used in this study, the transgene eGFP-L10a is regulated by the endothelial-specific VE-cadherin promoter. Isolation of eGFP-tagged-polysomes was performed by using anti-GFP-bound Dynabeads (Qiagen). After extensive washing, mRNA of endothelial cells from B16-F10 or HCmel12 tumors were obtained by Trizol and further purified by RNeasyMicro kits (Qiagen). Two different RNA pools, 1) the endothelial specific mRNAs of the tumor that were about to be translated and 2) a pool of unbound fraction (flow-through that did not bind to the anti-GFP beads), consisting of the rest of RNAs of the tissue, were collected.
3
Native ChIP Assay for H3K9me3
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Native ChIP assays were performed as described previously3 (link). A mouse monoclonal antibody against H3K9me3 (2F3) was used56 (link).
The antibody was incubated with anti-mouse IgG Dynabeads (Veritas for 1 h on ice and overnight after the addition of MNase-digested chromatin. For crosslinked ChIP, 1 × 107 cells were crosslinked with 1% formaldehyde at 25 °C for 10 min. Chromatin was extracted and then sonicated using Bioruptor USD-250 to obtain an average fragment size of 300–500 bp. Immunoprecipitation was performed using Dynabeads with antibodies, followed by purification using QIAquick PCR Purification Kit (Qiagen).
The antibody was incubated with anti-mouse IgG Dynabeads (Veritas for 1 h on ice and overnight after the addition of MNase-digested chromatin. For crosslinked ChIP, 1 × 107 cells were crosslinked with 1% formaldehyde at 25 °C for 10 min. Chromatin was extracted and then sonicated using Bioruptor USD-250 to obtain an average fragment size of 300–500 bp. Immunoprecipitation was performed using Dynabeads with antibodies, followed by purification using QIAquick PCR Purification Kit (Qiagen).
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