RNA Detection

Prepare fresh transfer buffer by adding 25 mL of 20transfer buffer and 50 mL of methanol to 425 mL of water. Following SDS-PAG ...

the antigens Mw. Cut out these boxes from the acetate film to create windows that locate protein–RNA signal (Fig.2a). Remove tr ...

Add 1μL of 10 mM dNTPs and 0.5μL of 0.5 pmol/μLRT# primer to each sample (seeNote 20). Denature RNA using the following thermoc ...

Assemble the XCell III gel system according to manufacturer’s instructions using a 6% TBE-UREA gel (seeNote 21) and fill chambe ...

Cut size: Insert size: (a) Low band 70–80 nt 18–28 nt (b) Medium band 80–100 nt 28–48 nt (c) High band 100–150 nt 48–98 nt Tran ...

Resuspend cDNA pellet in 8μL of freshly prepared circulariza- tion mix and transfer to 0.2 mL PCR tubes. Incubate for 1 h at 60 ...

Important: Following the PCR, do not open PCR tubes in same room where iCLIP library preparations (i.e., all steps before step 8 ...

Band size: Insert size: (a) Low 145–155 nt 18–28 nt (b) Medium 155–175 nt 28–48 nt (c) High 175–225 nt 48–98 nt Optional: Steps ...

Prepare qPCR library quantification standards and nontem- plate controls as per manufacturer guidelines. In technical triplicat ...

iCLIP proteins. It is also recommended that inputs are normal- ized by carrying out protein quantification following lysis and d ...

digestion conditions [20]. In order to ensure samples are not over or under digested, it is critical that each new batch of RNas ...

extended periods on higher percentage precast gels in order to provide better resolution at these molecular weights. The high R ...

a later date if required. Should PCR analysis reveal inaccurate and high cutting, then the stored low band may also be pro- cess ...

Acknowledgments The iCLIP protocol described is based on previous versions devel- oped in the Ule and Konig labs by many individ ...

Biotechnol 29(7):607–614. doi:10.1038/nbt. 1873 Yeo GW, Coufal NG, Liang TY, Peng GE, Fu XD, Gage FH (2009) An RNA code for the ...

Chapter 30 RNA Tagging: Preparation of High-Throughput Sequencing Libraries Christopher P. Lapointe and Marvin Wickens Abstract ...

the cell using high-throughput sequencing. For applications involving mRNA networks, we commonly purify poly(A)- containing RNAs ...

425–600μm acid-washed glass beads. Phenol–chloroform–isoamyl alcohol (25:24:1) pH 6.7. Chloroform. 100% ethanol. 80% ethanol. T ...

RNA ISO Buffer: 0.2 M Tris–HCl pH 7.5, 0.5 M NaCl, 0.01 M EDTA, 1 v/v % SDS. Filter-sterilize before first use (0.2μm filter), ...

Second strand synthesis oligo 5^0 - GTTCAGAGTTCTACA GTCCGACGATCNNNNNN-3^0. The underlined portion corresponds to Illumina 5^0 a ...