Helicos™ RNAseq SamplePreparation
The following overview provides a summary of sample preparation for the
purpose of full transcriptome sequencing (RNAseq) on the HelicosTM Genetic
Analysis System. This protocol has been tested and optimized for 100 – 400 ng
of genomic DNA free, polyA+ RNA (although other starting amounts, higher or
lower, have also been used successfully). The total sample preparation time is
14 hours.
 Equipment needed:
o Bioanalyzer (to determine starting sample quality. Optional)
o Thermocycler
o Microcentrifuge (for 0.2 ml PCR tubes and for 1.5 ml tubes).
o Dynal® magnet
o NanoDrop spectrophotometer
o XCell SureLock® Mini-Cell CE mark gel electrophoresis system
(Invitrogen, EI0001 – or other equivalent).
 Reagents needed:
o RNeasy MinElute cleanup kit (Qiagen Cat# 74207; optional)
o 100% EtOH
o SuperScript III kit (Invitrogen, Cat #18080-051).
o RNase If (NEB, Cat# M0243L).
o Performa gel filtration columns (EdgeBio Cat# 42453).
o Terminal transferase (NEB Cat# M0315L)
o Helicos DGE assay reagent kit
o 4-20% TBE gel or similar (Invitrogen Cat# EC62252Box).
o 10X bluejuice Gel loading dye (Invitrogen Cat# 10816-015).
o 25bp ladder (Invitrogen Cat # 10597-011).
o SYBR Gold nucleic acid gel stain (Invitrogen, Cat # S11494)
o Biotin dideoxyATP (Perkin Elmer Cat# NEL548001EA)
o USER enzyme (NEB Cat # M5505L).
o Ampure beads (Agencourt Cat # 000130).
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 Reverse transcription thermocycler program:
65°C
4°C
15°C
25°C
40°C
55°C
85°C
4°C
5min
forever
forever
10 min
40 min
50 min
5 min
forever
1. Bioanalyzer (40 min.)
Run 1-5 ng of the sample on the Bioanalyzer to ensure it is not degraded
as fragmented or degraded RNA may be lost during RNA purification
(optional).
2. RNA purification. Recommended (20 min)
Purify and concentrate the RNA sample using the RNeasy MinElute
cleanup kit (Qiagen) according to the manufacturer’s instructions (this step
is optional).
3. RNA fragmentation. Recommended (5 min)
Heat fragment the RNA by placing it at 95oC for 5 min (this step will
improve evenness of coverage, but is not necessary).
4. cDNA synthesis (2.5 hours.)
Prepare first strand cDNA using the SuperScript III kit (Invitrogen) and
high concentration random hexamers:
a) Mix together:
RNA (100-400ng-bring up to
13ul in nuclease free water)
50ng/µl Random Hexamers
dNTP
Total
13 ul
10ul
2ul
25ul
b) Place the samples in the thermocycler and start the Reverse
Transcription thermocycler program. Allow the samples to incubate
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at 65oC for 5 min and place them directly on ice for 2 min. Move
the samples to room temperature.
c) Press ‘skip’ on the thermocycler to move to the 15oC forever step
d) Prepare the following mix:
10X buffer
0.1M DTT
25 mM MgCl
Total
5ul
5ul
10 ul
20 ul
e) Add 20ul of the mix to each tube.
f) Return the samples to the thermocycler and allow them to incubate
at 15oC for 20 minutes.
g) Remove the samples from the thermocycler to room temperature.
h) Add to each tube:
RNaseOut
SSIII
Total
2.5ul
2.5ul
5ul
i) Press skip on the thermocycler and allow it to reach 25oC before
placing the tubes back in. Let the program continue to completion
(25oC 10 min, 42oC 40 min, 55oC 50 min, 70oC 10 min, 4oC
forever).
5. RNA removal (30 min)
Add 1ul of RNaseH and 1ul of RNase If to each tube and incubate at 37oC
for 30min.
6. Sample purification (20min)
Purify twice using the Performa dye removal columns and quantify by a
UV spectrophotometer:
a) Spin the Performa column at 700 X g for 2 min.
b) Move the cartridge to a new collection tube.
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c) Add the sample onto the gel matrix without touching it.
d) Spin at 700 X g for 2 min. The eluate contains the sample.
e) Repeat steps 1-4 again with a fresh column for a second filtration.
f) Quantify on the NanoDrop.
7. Poly-A tailing (2 hours)
The polyA tailing step adds a 3’ poly-A tail to the sample, in the presence
of a tailing oligo carrier, using terminal transferase. PolyA tailing facilitates
hybridization to the flow-cell’s channel and sequencing.
PolyA tailing thermocycler program:

95°C
4°C
42°C
70°C
4°C
5 min
forever
1 hour
10 min
forever
a) Prepare the following mix:
100 ng cDNA
Oligo A (see DGE protocol)
Water
Total
Sample
Xul
5ul
28.5-Xul
33.5ul
Control
--------5
28.5
33.5ul
b) Place the tubes in the thermocycler and start the polyA tailing program.
Incubate at 95oC for 5 min, then place directly on ice for 2 min.
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d) Prepare the following mix (mix extremely well):
CoCl
10X TdT buffer
dATP
Terminal transferase
Total
5ul
5ul
5ul
1.5ul
16.5ul
e) Add 16.5 ul of the mix to the sample and mix very well by pipetting up
and down. Collect the reaction at the bottom of the tube by brief
centrifugation.
f) Place the samples back in the thermocycler and press ‘skip’ to continue
the polyA tailing program to completion (42oC 1 hour, 70oC 10 min, 4oC
forever).
8. Poly-A tailing assessment (1.5 hours)
Run 5 ul of the sample on a polyacrylamide gel using SYBR Gold staining
to verify the success of the tailing reaction (see the DGE protocol,
determining the success of the tailing reaction).
9. 3’ blocking and tailing oligo removal (2 hours)
1. 3’ Blocking
Block the sample’s 3’ end using biotin-ddATP and terminal transferase:
a) Denature the sample at 95oC for 5 min and place them directly on ice
for 2 min.
b) Add 0.3ul of biotin-ddATP
c) Add 1.5ul of terminal transferase
d) Incubate at 37oC for 45 min.
e) Heat denature at 70oC for 10 min
2. Tailing oligo carrier removal
Digest the tailing oligo using the USER enzyme:
a) Add 1 ul USER enzyme reagent to each sample.
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b) Incubate at 37oC for 30 min.
10. Sample purification (1.5 hours)
Purify using the AMPure kit (Agencourt)
a) Bring the AMPure beads to room temperature
b) Bring the sample volume up to 60 ul with water.
c) Mix the AMPure beads well by vortexing.
d) Add 72ul AMPure beads to the sample.
e) Incubate at room temperature for 30 min (flick the tube every 10
minutes to mix).
f) Collect the sample at the bottom of the tube by brief centrifugation and
place the reaction on the Dynal® magnet. Allow the beads to gather at the
side of the tube for 5min.
g) Prepare fresh 70%EtOH (500ul per sample).
h) Carefully remove the supernatant from the AMPure beads.
i) Leaving the beads on the magnet, wash them twice with 200ul 70%
EtOH.
j) Remove any visible trace of EtOH (brief centrifugation can help collect
EtOH drops at the bottom of the tube so that they can be removed).
k) Leave the tubes open for 5-7 min (the beads should appear dry, no
liquid drops should be visible in the tubes).
l) Resuspend the beads in 20 ul water (by pipetting up and down ~20
times).
m) Leave the tubes open for 30 min on the magnet.
n) Collect the supernatant from the beads and place in a new 1.5ml tube
(the supernatant contains the sample).
o) Resuspend the beads again in 20 ul of water, close the tubes and place
on the magnet for 5 min.
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p) Collect the (sample containing) supernatant and add to the first 20 ul.
q) The sample is now ready to be measured by the HelicosTM OptiHybTM
Assay.
11. Determine the sample concentrations based on the OptiHyb assay.
Hybridize to the Heliscope flow-cell at a loading concentration of 350pM.
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