Here at Lexogen we are focused on the short-read RNA sequencing workflow. While there are exceptions to this, the general outline of an experimental procedure is as follows:
➊ RNA is isolated from the sample and contaminating DNA is removed, e.g., with DNase treatment.
➋ If needed for the method, the RNA is pre-treated, i.e., the mRNA is enriched by poly(A) selection or rRNA depletion.
Very commonly a 3’ mRNA library generation method is used which enriches for poly(A) tail-containing material, as this region is rich in features and enables sound differential gene expression studies without over-working the samples ahead of time.
➌ RNA can then be fragmented. If using fragmentation-free protocol, this point can be ignored.
➍ Reverse transcription is performed using the RNA sample and library generation primers as chosen.
➎ A second strand synthesis is then performed by randomly priming along the first cDNA strand.
➏ At this point the libraries are ready for end repair and the ligation of any products occurs on either double stranded cDNA.
➐ PCR is finally performed to add indices to the library, and / or amplify the material for library quality control.
The individual steps to prepare NGS libraries and the molecular principles underly these reactions are described in more detail in Chapter 7: RNA-Seq Library Preparation – Molecular Biology Basics.
Lexogen library preps for mRNA-Seq and whole transcriptome sequencing use a slightly different approach. Partial adapters are introduced already in the first step, e.g., during reverse transcription. Thereby, workflows are streamlined and efficiently shortened by removing various processing steps. This saves valuable time and allows to complete the library preparation workflow within a few hours. Figure 1 illustrates the schematic workflow for two different RNA-Seq library preparation methods.
RNA-Seq can be used for quite a variety of applications, is highly customizable by the combination of different methods, and offers sheer endless possibilities for modifications to fit the individual needs of a research project. Planning your experiment before taking up the pipets is the key to success and ensures high quality results to foster your ideas and hypotheses.
Figure 1 | RNA-Seq Library Preparation Workflows for Illumina short-read sequencing. RNA is extracted from cells, tissues, bioliquids or other samples and treated with DNase to remove genomic DNA prior to depletion of ribosomal RNAs or poly(A) RNA enrichment.