Journal: Nature methods

Article Title: Protein-RNA networks revealed through covalent RNA marks

doi: 10.1038/nmeth.3651

Figure Lengend Snippet: The RNA Tagging approach. a) Strategy. RBP, RNA-binding protein. PUP, poly(U) polymerase. b) Schematic of targeted RT-PCR and transcriptome-wide RNA Tagging assays. RNAs are tailed with a combination of guanosines (G) and inosines (I) (purple). The U-select primer contained the Illumina 3′ adapter sequence (brown), nine cytosines (purple) that base pair with the G/I tail, and three adenosines (red) that select for uridines at the 3′ end of the mRNA. c) Computational identification of Tagged RNAs. A-tails refers to the poly(A) tail and U-tails refers to 3′ terminal uridines, which were often in the U-tag. d) Nature of the data. The cartoon depicts Tagged RNAs aligned to a representative gene. ORF, open reading frame. e) Plot of the mean U-tag length detected by high-throughput sequencing of synthetic DNA libraries that contained U-tags of 0, 2, 4, 6, 8, 10, and 12 nucleotides. At least 50,000 reads were detected for each library (>1 million total reads). The R 2 value ( R 2 = 0.99, n = 7) was determined by linear regression analysis, and error bars represent standard deviation.

Article Snippet: 60 μL of cDNA was added to 10 μL of 10X Klenow Buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCL 2 , 10 mM DTT, 0.5 mg per mL BSA), 12 μL of water, 5 μL of 10 mM dNTPs, 10 μL of 10 μM 2 nd strand synthesis primer (GTTCAGAGTTCTACAGTCCGACGATCNNNNNN), and 3 μL of 5 U per μL Exo- Klenow DNA Polymerase (Life Technologies).

Techniques: RNA Binding Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Next-Generation Sequencing, Standard Deviation