By Ulf Andersson Ørom
This quantity presents a complete evaluation of the experimental and computational methodologies used to check the functionality of lengthy non-coding RNA (ncRNAs) expressed from enhancers. bankruptcy element either wet-lab and dry-lab options and annotating lengthy ncRNAs and exploring transcription by means of assessing the place transcription starts off and usually the way it occurs.Written within the hugely profitable Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and fending off recognized pitfalls.
Authoritative and state-of-the-art, Enhancer RNAs: equipment and Protocols goals to make sure profitable ends up in this swiftly constructing field.
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Additional resources for Enhancer RNAs: Methods and Protocols
Store at room temperature. 9. 5, 200 mM NaCl, and 2 % SDS. 10. 12 M urea, 10 mM EDTA, and 10× Denhardt’s solution. 11. 025 % sodium deoxycholate, 10 mM DTT, and 20 U/ml SUPERase-In. Make fresh. 12. 5 % SDS and 5 μg/μl proteinase K. Make fresh. 3 Reagents and Consumables 1. RNaseZap. 2. DEPC. 3. Nuclease-free H2O. 4. Phosphate-Buffered Saline (PBS). 5. 37 % Formaldehyde solution. 6. 20 U/μl SUPERase-In. 7. cOmplete EDTA-free protease inhibitor cocktail. 8. 1 U/μl RQ1 RNase-Free DNase. 9. 20 μg/μl Proteinase K solution RNA grade.
Wiley Interdiscip Rev RNA 1(2):266–286 5. Kim TK, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465(7295):182–187 6. Schaukowitch K, Joo JY, Liu X et al (2014) Enhancer RNA facilitates NELF release from immediate early genes. Mol Cell 56(1):29–42 7. Lai F, Orom UA, Cesaroni M et al (2013) Activating RNAs associate with mediator to enhance chromatin architecture and transcription. Nature 494(7438):497–501 8. Ule J, Jensen K, Mele A, Darnell RB (2005) CLIP: a method for identifying protein-RNA interaction sites in living cells.
14. Centrifuge at max speed for 10 min at 4 °C and then discard EtOH. 15. Let RNA pellet air dry. 16. Resuspend in 10–20 μL nuclease-free water. 5 Detecting RNA (See Note 4) 1. Treat 10 μL of RNA with 1 μL of DNaseI and incubate for 20 min at 37 °C. 2. Reverse transcribe RNA using a reverse transcription kit, with a final reaction volume of 20 μL. 3. Dilute cDNA 1:2. 4. Perform RT-qPCR using primers targeting your RNA of interest. Use 2 μL of diluted RT product in a 10 μL reaction volume. All RT-qPCR products should be checked by running on a 6 % PAGE gel for the expected sizes.