Yesterday I ordered all the reagents that I need to prep the libraries, I'm super excited about it especially since some arrived this morning. I'll be using a protocol designed by Sultan et al. (Sultan 2012) which maintains the strand specificity of the sequences. This is important because most imprinted transcripts lie in clusters in the genome (Edwards 2007; Reik 1997) which seem to be more gene-dense than the rest of the genome and can have overlapping transcripts on the + and - strands (Ideraabdullah, 2008). Maintaining strand-specificity will allow me to keep overlapping genes separate in my analysis.
The Sultan (2012) protocol to maintain strand specificity is pretty slick. Here's how it goes:
1. Extract total RNA (tRNA). I'll use an Omega miRNA kit for this and also a genomic DNA kit (in case I want to go back later and look at methylation patterns of the DNA). As placenta is a fairly heterogeneous tissue I need to use the entire sample to assure an even sampling of each of the three main layers. This means that I need to collect archival quality DNA and RNA as I won't be able to go back later if I decide I need something extra. I will grind it with liquid nitrogen and take some of the homogenate for the RNA kit, and some for the DNA kit.
2. Purify the tRNA. There are many types of RNAs in a tissue. I am only interested in messenger RNAs (mRNA) which will all have a poly-A tail. To purify the mRNA from tRNA I will use magnetic beads that have a poly-T oligo attached to them. These will bind to the mRNAs, magnetically bind to the plate, and keep my mRNA from being washed away with all the rest of the material. Then I will elute the mRNA away from beads and collect it for the next step.
3. Fragment the mRNA. mRNAs are much to long to sequence on the Illumina machine, so I need to break them up. I'll use a solution that Illumina provided to fragment the RNA.
4. First-strand synthesis. We do not have the technology to sequence RNA and so to get around this, we instead convert the RNA into DNA and then sequence that. DNA that came from RNA is called complement DNA (cDNA). As DNA is double stranded, this takes two steps, the first strand synthesis (with a reverse-transcriptase) and the second strand synthesis (with a regular polymerase).
5. Second Strand synthesis. This is the first step where the Sultan (2012) protocol differs from the standard Illumina Hiseq. Sultan (2012) calls for using dUTP instead of dTTP when forming the second DNA strand. This way I can later use an enzyme which chops up DNA on a "U" to cut away the second strand, leaving only the actual strand I am interested in - super clever.
6. End repair. After the mRNA-->cDNA conversion there are a lot of overhangs. Here I chop off all the extra so that each fragment has blunt ends.
7. Adenylate 3` end. In order to add on the adapters (necessary for the Illumina machine can sequence my cDNA library) I need an overhanging "A" on each 3` end. Here I add that "A".
8. Adapter ligation. Here I will barcode and add the adapters onto each of my sequences. I'll be splitting my 40 samples into 2 lanes of HiSeq, so I need 20 different barcodes. Here I will also have to make sure and not introduce batch effects into my design (Auer, 2010). I'll probably have another post entirely about this.
9. U-digestion. Here I will digest out the cDNA with "U"s in it so that I only have DNA oriented in the correct way. I will of course amplify this DNA, but it will cause the adapters to be wrong for the Illumina platform, so while they will be present, they won't be sequenced. Clever, clever, clever.
10. Enrich DNA fragment. Here I will PCR the library so that I have many copies of each molecule. This way there will be plenty for the Illumina machine to sequence.
11. Pool libraries. I will combine 20 of the samples into one lane and the other 20 for the other lane. Then send them off for sequencing.
So in short, I can keep almost the entire Illumina HiSeq kit as per usual, with a couple minor changes to maintain strand specificity.
As you may have noticed my in-text citations, it's because I recently bought the program papers, and am now testing it out in Chrome. It's nice by the way, especially for pages which I've recently started using. However, it looks like it won't format the bibliography directly from in-text citations in chrome, though it will format them properly if you go through and select each reference you want to work with.
Auer, P. L. and R. W. Doerge. 2010. Statistical Design and Analysis of RNA Sequencing Data. Genetics 185:405–416.
Ideraabdullah, F. Y., S. Vigneau and M. S. Bartolomei. 2008. Genomic imprinting mechanisms in mammals. Mutat. Res. 647:77–85.
Edwards, C. A. and A. C. Ferguson-Smith. 2007. Mechanisms regulating imprinted genes in clusters. Current Opinion in Cell Biology 19:281–289.
Reik, W. and E. R. Maher. 1997. Imprinting in clusters: lessons from Beckwith-Wiedemann syndrome. Trends in Genetics 13:330–334.
Sultan, M., S. Dökel, V. Amstislavskiy, D. Wuttig, H. Sültmann, H. Lehrach and M.-L. Yaspo. 2012. A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods. Biochemical and Biophysical Research Communications 422:643–646. Elsevier Inc.
No comments:
Post a Comment