1. mRNA Sequencing Sample Preparation
1. Quality check of total RNA
Customers should carry out a quality check of
their total RNA by running it out on a 1% agarose gel,
and the integrity of RNA judged upon staining with
ethidium bromide.
High quality, intact RNA will show a 28S rRNA band at
4.5kb, that should be about twice the intensity of the
18S rRNA band at 1.9kb. Both kb determinations
are relative to a 1kb ladder. The mRNA will appear as
A smear from 0.5-6kb.
Completely degraded RNA will appear as a very low
molecular weight smear.
Customers are to supply 10ug of purified total RNA

2. mRNA Sequencing Sample Preparation
2. mRNA Purification from Total RNA
mRNA is isolated from total RNA by binding the mRNA to a magnetic oligo(dT) bead. mRNA has a polyA tail and will bind to the oligo(dT) bead.
mRNA
5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
Magnetic
Oligo (dT)
bead
Total RNA containing
mRNA is added to
magnetic oligo (dT)
beads
PolyA Tail
3’-TTTTTTTTTTTTT
5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
3’-TTTTTTTTTTTTT
mRNA is then eluted
from the magnetic oligo
(dT) beads by heating to
800C for 2 minutes, & transferred
in the supernatant to another tube
mRNA in supernatant
5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’

3. mRNA Sequencing Sample Preparation
3. Fragmentation of mRNA
The mRNA is fragmented into small pieces using divalent cations under elevated temperature.
5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
mRNA
Add fragmentation buffer and PCR thermocycle
At 700C for 5 minutes
Random fragmentation 5’ 3’ 5’ 3’ 5’ 3’
Generates fragments ranging in size
from 100 bases to 5000 bases. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
4. First Strand cDNA Synthesis
Random Hexamer
Primers
The cleaved RNA fragments are copied
into first strand cDNA using reverse transcriptase
and a high concentration of random hexamer primers
mRNA 5’ 3’ 3’ 5’
cDNA
strand

4. mRNA Sequencing Sample Preparation
5. Second Strand cDNA synthesis
Remove the strand of mRNA and synthesize a replacement strand generating double-stranded cDNA.
mRNA
RNase H
5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
First Strand
cDNA
3’-AGCCTTCGACTTCACTAGTCCCAAGTTATTTTTTTTTTTTT-5’
RNase H activity enzymatically degrades the mRNA strand.
3’-AGCCTTCGACTTCACTAGTCCCAAGTTATTTTTTTTTTTTT-5’
DNA Polymerase I activity generates second strand cDNA
to form double-stranded cDNA.
DNA Polymerase I
Second Strand
cDNA
5’-TCGGAAGCTGAAGTGATCAGGGTTCAATAAAAAAAAAAAAA-3’
3’-AGCCTTCGACTTCACTAGTCCCAAGTTATTTTTTTTTTTTT-5’
First Strand
cDNA

5. mRNA Sequencing Sample Preparation
6. Add ‘A’ Bases to the 3’
End of the DNA Fragments
P5’-AGTCTTGGATCGAC-3’
3’-TCAGAACCTAGCTG-5’P
An ‘A’ base is added to the 3’ end of the
blunt phosphorylated DNA fragments, using
the polymerase activity of Klenow fragment
(3’ to 5’ exo minus). This prepares the DNA
fragments for ligation to the adapters, which
have a single ‘T’ base overhang at their 3’ end
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P
7. Ligate Adapters to the DNA
Fragments
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P
Adapters are ligated to the ends of the
DNA fragments, preparing them to be
Hybridized to the flow cell
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P

6. mRNA Sequencing Sample Preparation
8.Purify Ligation Products
The products from the ligation are purified on a
2% agarose gel, to remove all unligated adapters,
remove any adapters that may have ligated to one
another, & select a size range of templates to go on
The cluster generation platform
Excise a gel slice in the 200bp (+/- 25bp) and purify

7. mRNA Sequencing Sample Preparation
9. Enrich the Adapter-Modified cDNA Fragments by PCR
PCR is used to selectively enrich those cDNA
fragments that have adapter molecules on both
ends, & to amplify the amount of cDNA in the
library. The PCR is performed with two primers
that anneal to the ends of the adapters
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P
Denaturation
P5’-AGTCTTGGATCGACA-3’
Primer Annealing
Amplify using the following PCR protocol:
*30 seconds at 980C
*15 cycles of:
10 seconds at 980C
30 seconds at 650C
30 seconds at 720C
*5 minutes at 720C
*Hold at 40C
3’-ATCAGAACCTAGCTG-5’P
Extension
P5’-AGTCTTGGATCGACA-3’
3’-NNNNNTCAGAACCTAGCTGTNN-5’
5’-NNTAGTCTTGGATCGACNNNNN-3’
3’-ATCAGAACCTAGCTG-5’P

8. mRNA Sequencing Sample Preparation
10. Validate the Library
Perform the following quality control steps on the DNA
Library:
Determine the library concentration by measuring its
absorbence at 260nm. The yield from the protocol
should be between ng of DNA.
Measure the 260/280 ratio. It should be ~
Either load 10% of the volume of the library on a gel
and check that the size range is as expected, or run the
DNA library on an Agilent 2100 Bioanalyzer.
Sample
Determine the molar concentration of the library ready for Cluster Generation
Data from Agilent 2100 Bioanalyzer