RNA Detection

Chapter 3 Identifying the m 6 A Methylome by Affinity Purification and Sequencing Phillip J. Hsu and Chuan He Abstract N^6 -meth ...

m^6 A-seq consists of affinity purification of m^6 A-marked RNA fragments using an m^6 A-specific antibody, followed by high- th ...

RNeasy Plus Mini Kit (Qiagen,seeNote 1). mRNA Miniprep Kit (e.g., Sigma-Aldrich GenElute). Gel Extraction Kit (e.g., Qiagen Min ...

Elution buffer (100μL per reaction, kept on ice, prepared fresh): 20μLof5IP buffer, 1μL of SUPERase inhibitor, 33 μLof20mMm^6 ...

Isolate the RNA using the RNeasy kit following the manufac- turer’s protocol, being sure to use the gDNA Eliminator Col- umns t ...

Add 0.1 volumes (10μL) of 3 M sodium acetate to the com- bined eluents, followed by 2.5 volumes (275μL) of 100% ethanol. Mix we ...

amplification curve calculated in the previous step. Adjust the amount of Input and IP cDNA template so that the same number of ...

as HEK293T, pipette the ice-cold PBS gently onto the edge of the cell culture dish when washing to minimize loss of cells. Work ...

It is recommended to perform the PCR reaction at half scale, saving half of the template in case more or fewer PCR cycles are n ...

Chapter 4 PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution Zhipeng Lu, Jing Gong, ...

sequence, structure, expression pattern, RNA–protein complex compositions, and functions [5–8]. A detailed description of the ph ...

also discussed some alternative solutions to tailor the analytic method for specific usage. Several of the library preparation s ...

20% N-Lauroylsarcosine sodium salt solution (e.g., Sigma- Aldrich). Deoxycholic acid sodium salt (e.g., Thermo Fisher Scientifi ...

P3-Solexa PCR primer (PAGE purified): 5^0 -CAAGC AGAAG ACGGC ATACG AGATC GGTCT CGGCATTCCT GCTGA ACCGC TCTTC CGATC T-3^0. P6-Sol ...

10 w/v % Sodium deoxycholic acid solution (NaDOC): Mix 5 g of deoxycholic acid sodium salt in 50 mL of nuclease-free water. Sto ...

254 nm and 365 nm emission UV bulbs (e.g., Agilent Technologies). Gel imager (e.g., Bio-Rad Gel Doc XR+). 300 nm blue light tra ...

3 Methods The following is a detailed description of the PARIS method (Fig.1). Indicators of successful implementation are inclu ...

The library preparation part (Subheadings 3.5 and 3.6) was adapted from a similar protocol for icSHAPE and modified [29]. The pr ...

break up and resuspend in urea/SDS solution as the DNA is fragmented by UV 365 nm in the presence of AMT. To the lysed cells ad ...

Note 5). Each ShortCut reaction in 50μL volume includes 20 μg RNA, 4μL ShortCut RNase III, 5μL 200 mM MnCl 2 , and 5μL ShortCut ...