mRNA Sequencing Sample Preparation

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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
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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
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mRNA Sequencing Sample Preparation
3. Fragmentation of mRNA
The mRNA is fragmented into small pieces using
divalent cations under elevated temperature.
mRNA
5-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3
Add fragmentation buffer and PCR thermocycle At
700C for 5 minutes
Random fragmentation
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3
5
3
5
3
Generates fragments ranging in size from 100
bases to 5000 bases.
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5
5
5
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5
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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
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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
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mRNA Sequencing Sample Preparation
6. Add A Bases to the 3 End of the DNA
Fragments
P5-AGTCTTGGATCGAC-3 3-TCAGAACCTAGCTG-5P
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-5P
7. Ligate Adapters to the DNA Fragments
P5-AGTCTTGGATCGACA-3 3-ATCAGAACCTAGCTG-5P
Adapters are ligated to the ends of the DNA
fragments, preparing them to be Hybridized to the
flow cell
P5-AGTCTTGGATCGACA-3 3-ATCAGAACCTAGCTG-5P
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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
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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-5P
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-5P
Extension
P5-AGTCTTGGATCGACA-3
3-NNNNNTCAGAACCTAGCTGTNN-5
5-NNTAGTCTTGGATCGACNNNNN-3
3-ATCAGAACCTAGCTG-5P
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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 500-1000ng of DNA. Measure the 260/280
ratio. It should be 1.8-2.0. 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