RNA-Seq Library Preparation | RNA Lexicon
lexogen.com/rna-lexicon-rna-seq-library-preparation-molecular-biology-basics
Lexogen August 23, 2021
After you have successfully extracted the RNA from your sample, controlled the quality of
your preparation, and removed residual gDNA (if needed), it is time to prepare your RNA-
Seq libraries. Depending on the library method you have chosen and the RNA fraction
you are interested in, you may need to pre-select for your RNA fraction of choice. For
more details on this, check out our chapter on RNA Enrichment and Depletion.
While the individual reaction steps in an RNA-Seq workflow can vary depending on the
library preparation method used, the molecular principles underlying these reaction steps
remain the same. The following reactions are commonly used in RNA-Seq library
preparation: Reverse Transcription, Second Strand Synthesis, End Repair, Ligation, and
PCR Amplification. In the following chapter, we will go over these steps and shed light on
the molecular basis of these reactions.
1. Reverse Transcription / First Strand Synthesis
First strand synthesis refers to the generation of a complementary DNA molecule from an
RNA template by an enzyme called Reverse Transcriptase. Reverse transcriptases are a
viral RNA-dependent DNA-polymerases that transcribe RNA template molecules into
copy DNA (cDNA). The discovery of reverse transcriptases in the 1970s1,2 has
revolutionized molecular biology by short-circuiting Francis Cricks’ original dogma of
molecular biology which described a unilateral flow of genetic information from DNA →
RNA → Protein (Figure 1). This discovery was awarded with a Nobel Prize in 1975.
1/7
Figure 1 | Central Dogma of Molecular Biology.
The central dogma of molecular biology describes the flow of genetic information within
biological systems. DNA can either be replicated by DNA Polymerase (DNA → DNA) or is
transcribed into RNA by RNA Polymerases (DNA → RNA). RNA is a messenger
molecule that can be translated into proteins by Ribosomes (RNA → protein). Reverse
transcription describes the conversion of RNA into DNA (RNA → DNA) by reverse
transcriptase and is commonly used by viruses. RNA replication generates RNA from
RNA.
Today, reverse transcription is at the core of all RNA-Seq experiments and as such
remains one of the fundamental reactions in state-of-the-art molecular biology
applications.
A reverse transcription reaction in an RNA-Seq workflow requires three sub-steps: primer
annealing, cDNA synthesis (Fig. 2), and enzyme inactivation.
2/7
Figure 2 | Reverse Transcription generates cDNA from RNA template molecules. A)
Oligo(dT)-primed reverse transcription does not require rRNA depletion or mRNA
selection due to the primer annealing specifically to the 3’ poly(A) tail of mRNAs. B)
Random-primed reverse transcription uses pre-selected or depleted input RNA. Random
primers hybridize along the RNA template. Reverse transcriptase elongates the primers
and generates a complementary DNA copy.
2. RNA Template Removal and Second Strand Synthesis
After the first DNA strand is generated, many RNA-Seq workflows require the generation
of a double stranded DNA molecule. The product of the reverse transcription reaction is a
cDNA single strand that is paired with the initial RNA template strand. In order to
generate the second DNA strand, the RNA first needs to be removed. There are different
ways to achieve RNA removal. Most commonly, the product is heated in a buffer that is
formulated to specifically hydrolyze RNA while the DNA strand stays intact.
Subsequently, random primers are annealed to the now accessible cDNA first strand and
the second strand is generated by incorporation of complementary nucleotides using a
DNA-dependent DNA Polymerase. For random primed second strand synthesis, DNA
polymerases that work at lower temperatures are used, while their thermostable
counterparts are mostly used in PCR amplification and for targeted RNA-Seq
approaches.
RNA template removal is required to ensure that short random primers used to initiate
second strand synthesis gain access to the cDNA first strand. In case a targeted
sequencing approach is used, RNA removal can be omitted. Targeted primers are
commonly designed with a high annealing temperature (>60 °C) to avoid unspecific
priming to non-target sequences. Concomitantly, the reaction is carried out at a much
higher temperature than random primed second strand synthesis. This elevated
3/7
temperature of 60 – 72 °C weakens the RNA-DNA interactions sufficiently for the targeted
primer to anneal to the cDNA first strand. The RNA template is unwound and the
complementary DNA second strand is generated.
Most DNA-polymerases used for second strand synthesis possess proofreading activity
and thus the error rates in this step are much lower than during reverse transcription.
The properties of natural reverse transcriptases in combination with a process called nick
translation for second strand synthesis were already exploited in the 1980s for efficient
generation of cDNA libraries3.
3. End Repair
First and Second Strand Synthesis generate partially double stranded DNA with single-
stranded ends. A process termed end repair is therefore often used to prepare the
double-stranded DNA library for the adapter ligation step. During the end repair reaction,
partial single strands on the 5’ end of the fragment are filled in by a polymerase to
generate a double strand using the protruding end as a template. Single-stranded 3’
overhangs on the other end of the DNA fragment are removed using a 3’ → 5’
exonuclease. This process creates blunt ends on both sides of the fragment.
Depending on the adapter ligation strategy, a single adenine can be added at the 3’ end
of each strand in a process referred to as A-tailing. These A-tailed fragments are
subsequently ligated to adapters with a single 5’ T-overhang in the subsequent ligation
step (Fig. 3).
As the efficiency of blunt end ligation is usually lower than ligation with overhangs (even if
it is just one nucleotide), end repair including A-tailing is a common theme in library
preparation for Next Generation Sequencing.
As DNA ligases require molecules with a 5’ phosphate (5’-P) and a 3’ hydroxyl group (3’-
OH) as substrates, these functional groups are also generated during the end repair
process. This is achieved either by using enzymes that generate such end products or by
enzymatic activities that transfer these functional groups, e.g., Polynucleotidekinase
(PNK) can transfer phosphate groups to the 5’ end of RNA.
There are also other variations of “end repair” used in RNA-Seq library preparation. The
common theme is that overhangs are removed or filled in. This is mostly done on DNA,
but RNA can also undergo end repair.
4. Ligation
We already outlined a few basic principles of ligation in the previous section, here we will
go into a few more details. Ligation in molecular biology refers to the enzymatic process
of joining two nucleic acid molecules by attaching the 3’-OH group of the first molecule to
4/7
the 5’-P group of the second molecule. Ligases were discovered by multiple labs4 in the
1960s and are also considered as one of the major breakthroughs in molecular biology,
enabling molecular cloning of recombinant DNA molecules and NGS library generation.
As mentioned above, ligation occurs in various modes.
Figure 3 | End Repair, A-tailing and TA ligation. After first and second strand synthesis,
single stranded overhangs are removed and the 3’ ends are adenylated. Pre-annealed
partially double-stranded sequencing adapters with 3’ T-overhangs are ligated to the A-
tailed inserts. Sequencing adapters consist of a P7 and a P5 linker sequence.
5. PCR Amplification
PCR or polymerase chain reaction is a widely used method to generate millions of DNA
copies from as low as a single molecule. The description of the PCR reaction5 and its first
application in the 1980s is mainly attributed to Kary Mullis who received the 1993 Nobel
Prize in Chemistry for his discoveries. The extreme sensitivity and versatility of PCR led
to its numerous applications in various fields of science ranging from molecular biology
research to medical diagnostics, to infectious disease detection (e.g., SARS-CoV-2
diagnostics), even down to paternity testing and criminal forensics, and has helped to
unravel the genome of Neanderthals.
5/7
PCR cyclers for running three different PCR reactions in one machine.
Thus, polymerase chain reaction is an integral part of cutting-edge science and used in
many state-of-the-art techniques, including high-throughput Next Generation Sequencing.
PCR is the final step in most NGS library preparation workflows. During this step, the
libraries are amplified for quality control. In case partial adapters were introduced in the
library generation step, the adapter sequences are completed and indices are introduced.
PCR uses a thermostable polymerase to amplify a DNA template by repeating three
steps in multiple cycles: denaturation, primer annealing, and elongation.
Literature:
1
Temin HM, Mizutani S (1970) RNA-dependent DNA polymerase in virions of Rous sarcoma
virus. Nature 226(5252):1211–1213
2 Baltimore D (1970) RNA-dependent DNA polymerase in virions of RNA tumour
viruses. Nature 226(5252):1209–1211
3 Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA
libraries. Gene 25(2-3):263-269
4 Lehman IR. DNA ligase: structure, mechanism, and function. Science. 1974 Nov 29;186(4166):790-
7. doi: 10.1126/science.186.4166.790. PMID: 4377758
5 Mullis, K.F.; Faloona, F.; Scharf, S.; Saiki, R.; Horn, G.; Erlich, H. (1986). “Specific enzymatic
amplification of DNA in vitro: The polymerase chain reaction”. Cold Spring Harbor Symposia on
Quantitative Biology. 51: 263–273. doi:10.1101/sqb.1986.051.01.032
Written by Dr. Yvonne Goepel
Are you ready to become an RNA Expert?
Sign up and gain access to helpful checklists in PDF format that can assist you in your
experiments. In addition, you’ll have the opportunity to download the RNA LEXICON E-
BOOK in PDF format as well.
6/7
SIGN UP
7/7
