Degradome sequencing

Degradome sequencing (Degradome-Seq), also referred to as parallel analysis of RNA ends (PARE), is a modified version of 5'-Rapid Amplification of cDNA Ends (RACE) using high-throughput, deep sequencing methods such as Illumina's SBS technology. The degradome encompasses the entire set of proteases that are expressed at a specific time in a given biological material, including tissues, cells, organisms, and biofluids. Thus, sequencing this degradome offers a method for studying and researching the process of RNA degradation. This process is used to identify and quantify RNA degradation products, or fragments, present in any given biological sample. This approach allows for the systematic identification of targets of RNA decay and provides insight into the dynamics of transcriptional and post-transcriptional gene regulation.

Degradome sequencing is a complex process which includes multiple steps such as isolating RNA fragments in a given sample as well as ligation and reverse transcription to form complementary DNA (cDNA) strands. This cDNA can be sequenced, and the results are compared with a transcriptome, or reference genome, in order to determine and characterize the abundance of the RNA fragments identified in this process.

Methods

Technique

In general, the basic steps necessary for degradome sequencing include:

RNA isolation and separation: The RNA is isolated from the sample and size-fractionated to separate and classify small RNA molecules.

Library sequencing: The RNA is then ligated and used to form cDNA strands via reverse transcription. The cDNA is then denatured and replicated via polymerase chain reaction (PCR).