An essential step in preparing total RNA or genomic DNA samples for sequencing is cutting them down into usable fragments. For Illumina instruments, the bridge amplification aspect of the sequencing process works best when these fragments are between 100 and 1000 nucleotides long, and quality is improved by using fragments within a narrow size range (ideally no more than 200bp spread). There are several different techniques for accomplishing this: nebulization, sonication, and enzymatic fragmentation.
For a DNA fragment to be sequenced on an Illumina instrument, it first has to attach to the Illumina flow cell. The interior of the fluidics lane on each flow cell is printed, top and bottom, with a lawn of single-stranded oligonucleotides. All our DNA of interest needs to have is a complementary region on each end for clustering and sequencing to take place.
mRNA – messenger RNA – is typically around 5% of the total RNA in a cell, and in most eukaryotic organisms the vast majority of it is tagged with a poly(A) tail. This allows these molecules to be isolated out from a total RNA sample, reducing the number of reads needed to sequence the transcriptome or analyze gene expression. Continue reading “Stranded mRNA Sequencing”
It’s always useful to have a method for determining the relative quality of one’s source material. If you’re starting with garbage, you’re unlikely to have anything but garbage at the end, no matter how well you execute your protocols or how skillfully you massage your data. For RNA, one of the primary means of determining quality is the RNA Integrity Number, or RIN score, developed by Agilent. Continue reading “What is a RIN score?”
The composition of RNA in a cell can be roughly estimated at 80% ribosomal RNA, 15% transfer RNA, and 5% messenger RNA. With total RNA sequencing, all of those subtypes (as well as any small RNAs) are converted into cDNA for sequencing. Continue reading “Total RNA Sequencing”