Week 6: Libraries (No Books Involved)

Hi Reader,

Thank you once again for returning to my blog post! Today we’re going to be talking about libraries. 

Scientific Overview 

A library is a collection of books. But, that’s not the kind of library we’re interested in right now. To sequence RNA, we need to create a library, or a collection of modified DNA molecules that the sequencing machine can read. Let’s go step by step through the diagram below which shows how a library is prepared.  

1. We start with total full-length RNA, which as you surely recall,  I extracted from the angelfish’s tissue. 
2. Next, we anneal primers or short nucleotide fragments near the ends of the RNA molecules. 
3. After annealing, we need to convert that RNA into DNA that the sequencing machine can read. At first, this might seem like it’s an impossible task. After all, the central dogma of molecular biology (Week 1 post!) clearly states that DNA can only be transcribed into RNA and RNA only translated into proteins. You can’t go “backwards” from RNA to DNA can you? Well actually you can!! Nature evolved a unique class of enzymes called reverse transcriptases. These reverse transcriptases come from a group of retroviruses with RNA as their genetic material. So in this step, the reverse transcriptase is added to the RNA for revere transcription. (Fun fact: The reverse transcriptase I used in this project was genetically engineered form the Moloney Murine Leukemia Virus.) 
4. After the RNA is reverse-transcribed into DNA also called “cDNA”, we need to amplify the DNA until we have enough for sequencing. To accomplish this we use PCR or Polymerase chain reaction.  In the PCR reaction, we combine the cDNA with raw nucleotides, and a heat stable polymerase. This polymerase assembled the raw nucleotides into identical copies of the cDNA, and for every PCR cycle, the amount of DNA we have doubles. Below are the steps of PCR. There is a denaturation step where double stranded DNA is separated into single strands, an annealing step where primers are bound to the template DNA, and then an elongation step where the polymerase builds an identical complementary copy of the DNA molecule. 
5. Finally, we attach 1D adapters that allow the new DNA to can now be loading into and read by the sequencing machine. 

That completes our overview of the library preparation process. 

My experimental process 

It took almost 7 hours of hands-on wet-bench work to complete the library preparation process. This requires focus and concentration. One mistake during the process means you have to start all over. Even worse, I only had enough reverse transcriptase to prepare the library once. If I messed up, I’d need to buy more, not only delayed me but also costing hundreds of dollars. Furthermore, you can’t actually know if your library preparation was fully successful until you actually start sequencing, so I was definitely going through this procedure blind. I started the library preparation with around 200ng of RNA and I ended up with 130 ng of cDNA. So there was a significant amount of loss through the library preparation process (which is expected).