MaTAR25 RNA Antisense Pulldown and Mass Spectrometry
UID: 10797
- Description
- Description from PRIDE: "It has been suggested that lncRNAs can interact with transcriptional regulators/co-factors to form ribonucleoprotein (RNP) complexes to regulate the expression of downstream genes in the cell nucleus. To identify potential interacting proteins of MaTAR25, we used two different paired sets of biotin-labeled antisense oligonucleotides targeting MaTAR25 for native RNA antisense oligonucleotide pull-down (RAP) in 4T1 cells followed by qRT-PCR to assess pull-down efficiency. Samples were also eluted from beads for mass spectrometry isobaric tags for relative and absolute quantitative (MS-iTRAQ) analysis to identify proteins that bind to MaTAR25, and PPIB as the corresponding control. We ranked the candidate interactors based on detectable peptides above background in both pair sets of oligonucleotide pull-downs, and selected candidates with at least 2-fold enrichment compared to corresponding PPIB oligo pull-down."
"Sample Processing Protocol: Cells were lysed in a 10 cm culture dish in 1 ml IP lysis buffer (IPLB, 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol, supplemented with 100 U/ml SUPERase-IN and 1X Roche protease inhibitor cocktail) for 10 minutes, and lysate was centrifuged at 13,000xg for 10 minutes. Cell lysate was adjusted to 0.3 mg/ml (Pierce BCA Protein Assay). A total of 100 pmol of biotinylated oligo was added to 500 μl of lysate and incubated at room temperature for 1 hour with rotation. 100 μl streptavidin Dynabeads were washed in IPLB, added to the lysate, and incubated for 30 minutes at room temperature with rotation. Beads were washed three times with 1 ml lysis buffer. For determining temperature for optimal elution, beads were then resuspended in 240 μl of 100 mM TEAB and aliquoted into eight PCR tubes. Temperature was set on a veriflex PCR block and incubated for 10 minutes. Beads were captured and TRIzol was added to the eluate and beads. Once optimal temperature is established, the beads were resuspended in 90 μl of 100 mM TEAB, and incubated at 50° C for 10 minutes. TRIzol was added to 30 μl of the eluate, another 30 μl was kept for immunoblots, and the last 30 μl aliquot was sent directly to the Cold Spring Harbor Laboratory Mass Spectrometry Shared Resource for analysis."
"Data Processing Protocol: Instrument type ESI-QUAD Mass values Monoisotopic iTRAQ® Reagents - 4plex Applications for quantification and ratio comparsion. Protein ratio type weighted Fragment mass tolerance 0.2 Da Significance threshold p< 0.00182"
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Access via PRIDE
RAW files of Proteome analysis available through FTP link or website download
Accession #: PXD017398Access via ProteomeXchangeRAW files of Proteome analysis available through FTP link or website download
Accession #: PXD017398 - Access Restrictions
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Free to All
- Access Instructions
- All public datasets available in PRIDE Archive in the repository can be accessed via the website by FTP link, Web Service (for programmatic access), and the PRIDE Inspector stand-alone tool.
- Associated Publications
- Data Type
- Equipment Used
- Dataset Format(s)
- RAW
- Data Tool(s)
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Mass Spectromety
- Dataset Size
- 4.3 GB
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