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1. Materials and Methods
This method involves several steps that are designed to convert total RNA into a library of
template molecules suitable for high throughput DNA sequencing. The first step involves
purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic
beads. Following purification, the mRNA is fragmented into small pieces using divalent
cations under elevated temperature. Then the cleaved RNA fragments are copied into first
strand cDNA using reverse transcriptase and a high concentration of random hexamer
primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and
RNaseH. Finally the short cDNA fragments are prepared for sequencing on the Illumina
Genome Analyzer using reagents provided in the Genomic DNA Sequencing Sample Prep
Kit available with the system. The details of all of these steps are as follows:
1.1
mRNA purification from Total RNA
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Dilute 10µg of total RNA with nuclease-free H2O to 50µL in a 1.5 mL RNase free
non-sticky Eppendorf tube.
Heat the sample at 65°C for 5 minutes to disrupt the secondary structures, and
place the tube on ice.
Meanwhile, aliquot 100µL of Dynal oligo(dT) beads (Invitrogen, #610-06) into a
1.5mL RNase free non-sticky eppendorf tube.
Wash the beads twice with 100µL of Binding Buffer (20mM Tris-HCl pH 7.5,
1.0M LiCl and 2mM EDTA), and remove the supernatant.
Resuspend the beads in 50µL of Binding Buffer, and add the 50µL of total RNA
sample from step 2; rotate the tube at RT for 5 minutes, and remove the
supernatant.
Wash the beads twice with 100µL of Washing Buffer B (10mM Tris-HCl PH 7.5,
0.15M LiCl, 1mM EDTA).
Prepare for second round of oligo-dT purification by aliquoting 80µL of Binding
Buffer to a fresh 1.5mL RNase free non-sticky Eppendorf tube.
Remove the supernatant from the beads of step 6, add 20µL of 10mM Tris-HCl
and heat the beads at 800C for 2 minutes to elute mRNA. Immediately put the
beads on the magnet stand and transfer the supernatant (mRNA) to the tube from
step 7. Add 100µL of Washing Buffer B to the remaining beads.
Heat the mRNA sample from step 8 at 65°C for 5 minutes to disrupt the
secondary structures, and place the tube on ice.
Meanwhile, wash the beads from step 8 twice with 100µL of Washing Buffer B,
and remove the supernatant.
Add 100µL of mRNA sample from step 9 to the beads; rotate the tube at RT for 5
minutes.
Remove the supernatant and wash the beads twice with 100µL of Washing Buffer
B.
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13.
Remove the supernatant from the beads, add 10µL of 10mM Tris-HCl and heat
the beads at 800C for 2 minutes to elute mRNA. Immediately put the beads on the
magnet stand and transfer the supernatant (mRNA) to a fresh 200µL thin wall
PCR tube, and there should be ~9µL of mRNA.
Note: you may also start with 100ng mRNA, and directly go to step 2.2.
1.2
Fragmentation of mRNA
1.
Assemble the following reaction:


2.
3.
4.
1.3
10 x Fragmentation Buffer (Ambion, #AM8740)
mRNA (100ng)
1 µL
9µL
Incubate the tube in a PCR thermocycler at 70°C for exactly 5 minutes, add 1 µL
of Stop Buffer (Ambion, #AM8740), and put the tube on ice.
Transfer the solution to a 1.5 ml microcentrifuge tube. Add 1 µL of 3M NaOAC,
pH 5.2, 2 µL of glycogen (5ug/µL, Ambion, #AM9510), and 30 µL of 100%
EtOH. Incubate the tube at -80°C for 30 minutes.
Spin the tube at 14000 rpm for 25 minutes at 4°C in a microcentrifuge. Wash the
pellet with 70% EtOH, and air dry the pellet. Resuspend the RNA in 10.5µL of
RNase free water.
First strand cDNA synthesis
1.
Assemble the following reaction in a 200uL thin wall PCR tube:


2.
3.
5.
1 µL
10.5µL
Incubate the sample in a PCR thermocycler at 65°C for 5 minutes, and then place
the tube on ice.
Mix the following in order, make 10% extra reagent for multiple samples:




4.
Random Hexamer Primers (3ug/µL, Invitrogen, #48190-011
mRNA (100ng/µL)
5 first strand buffer (Invitrogen, #18064-014)
100mM DTT (Invitrogen, #18064-014)
dNTP mix (10mM)
RNaseOUT (40U/µL) (Invitrogen, #10777-019)
4µL
2µL
1µL
0.5µL
Add 7.5µL of mixture to the tube, mix well, and heat the sample at 25°C in a
thermocycler for 2 minutes.
Add 1 µL SuperScript II (200U/ µL, Invitrogen, #18064-014) to the sample, and
incubate the sample in a thermocycler with following program:
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



1.4
Step 1
Step 2
Step 3
Step 4
25°C
42°C
70°C
4 °C
10 min
50 min
15 min
Hold
Second strand cDNA synthesis
1.
2.
3.
Put the tubes from section 2.3, step 5 on ice.
Add 61µL of H2O to the first strand cDNA synthesis mix.
Add the following reagents:

4.
5.
6.
10 second strand buffer (500mM Tris-HCl pH7.8, 50mM MgCl2, 10mM
DTT)
10µL
 dNTP mix (10mM)
3µL
Mix well, incubate on ice 5 minutes or until well chilled, and add:
 RNaseH (2U/µL, Invitrogen, #18021-014)
1µL
 DNA Pol I (10U/µL, Invitrogen, #18010-025)
5µL
Mix well, and incubate at 16°C in a thermocycler for 2.5 hours.
Purify the DNA with a QIAquick PCR spin column (Qiagen, #28106), and elute
in 30µL of EB solution.
Note: Steps 1.5 through 1.9 use reagents that are part of the Illumina
Genomic DNA Sample Prep Kit (part # 1000181), unless otherwise
indicated.
1.5
End repair
1.
Prepare the following reaction mix:






Eluted DNA
H2O
T4 DNA ligase buffer with 10mM ATP
dNTP mix (10mM)
T4 DNA polymerase (3U/µL)
Klenow DNA polymerase (5U/µL)
30µL
45µL
10µL
4µL
5µL
1µL
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
2.
3.
1.6
Incubate the sample at 20°C for 30 minutes.
Purify the DNA with a QIAquick PCR spin column (QIAGEN, #28106), and
elute in 32µL of EB solution.
Prepare the following reaction mix:




2.
3.
Eluted DNA
Klenow buffer
dATP(1mM)
Klenow 3’ to 5’ exo- (5U/µL)
32µL
5µL
10µL
3µL
Incubate the sample at 37°C in for 30min.
Purify the DNA with a QIAquick MinElute column (QIAGEN, #28006), and
elute in 19µL of EB solution.
Adaptor ligation
1.
Prepare the following reaction mix:




2.
3.
1.8
5µL
Addition of a single <A> base
1.
1.7
T4 PNK (10U/µL)
Eluted DNA
DNA Ligase buffer
Adaptor oligo mix
DNA Ligase (1U/µL)
19µL
25µL
1µL
5µL
Incubate the sample at RT for 15min.
Purify the DNA with a QIAquick MinElute column (Qiagen, #28006), and elute
in 10µL of EB solution.
Gel purification of cDNA Templates
1.
2.
3.
Prepare a 2% agarose gel in a final volume of 50mL 1x TAE buffer.
Load the 10µL of sample into one well, and 1µL of 100bp DNA ladder
(Invitrogen) into another well. (for handling multiple samples, leave at least 2
wells between samples to prevent cross contamination)
Run gel at 120V for 45~60min till sufficient separation of the 100bp and 200bp
bands of the DNA ladder.
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4.
Cut a gel slice at 200 bp (+/- 25bp) as showing in figure 1, and purify the DNA
with QIAquick gel extraction kit (Qiagen, #28706). Elute the DNA into 30µL of
EB solution.
300 bp
200 bp
100 bp
Figure 1: A 2% Agarose gel showing the region that should be cut to purify DNA fragments for library
preparation.
1.9
PCR Enrichment of Purified cDNA Templates
1.
Set up PCR master mix, make 10% extra reagent for multiple samples, and
aliquot 20µL to each PCR tube:






5  cloned Phusion Buffer (NEB, #F-530)
PCR primer 1.1
PCR primer 2.1
25mM dNTP mix
Phusion polymerase (NEB, #F-530)
H2O
10µL
1µL
1µL
0.5µL
0.5µL
7µL
2.
Add 30µL of purified ligation mix from section 2.8, step 4 to the PCR tube.
3.
Run following PCR cycle:






98°C
98°C
65°C
72°C
72°C
4°C
30 sec
10 sec
30 sec
30 sec
5 min
hold
15
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4.
Purify the DNA with a QIAquick column (Qiagen, #28106), and elute in 30µL of
EB solution.
5.
Run 1 uL of the purified product from step 4 on an Agilent Bioanalyzer DNA
1000 chip (Agilent, #5067-1504) to QC the final product and quantify the DNA
concentration. Successful preparation should yield a distinct band at ~200 bp, as
shown in figure 2.
Figure 2: An image of the data generated from an Agilent Bioanalyzer showing the ~200 bp PCR product
produced from the protocol.