Determination of RNA Secondary Structure By RNA-Sequencing of HEK293T Cells

RNA molecule fold into three dimensional structures in order to fill important roles in the cell such as translation and post-transcriptional regulation. These three dimensional structures, in turn, depend on the pattern of hydrogen bonds between ribonucleotides. We employ a technique based on high throughput RNA Sequencing (RNA-Seq) to uncover this patter on a transcriptome wide scale. Overall design: We take advantage of two ribonucleases that have structure specific activity. RNAse S1 specifically digests single stranded RNA while RNAse V1 is specific to double stranded RNA. We aliquot each of our sample into to reactions. One is thoroughly treated with RNAse S1, digesting away single stranded RNA and leaving behind dsRNA. The other is treated with RNAse V1, which has the inverse effect. The surviving dsRNA and ssRNA molecules are then subject to high throughput sequencing. The resulting reads are used to generate a structure score, revelaing which regions of each transcript are involved in base pairing and which are unpaired.