cDNA SYNTHESIS FOR RNASeq LIBRARY
PREPARATION
This method is based on Illumina’s RNASeq library preparation protocol and
tested by Apratim.Chakrabarti. However, all these reagents can be prepared
or purchased from several vendors. Also, the work flow is suggestive only and
can be stopped at many points which are indicated. If you have further
questions
or
suggestions,
please
contact
Karen
Moore
(kamoore@exeter.ac.uk)
Consumables:
1. RNAse free water (DEPC treated MiliQ)
2. 10mM Tris-Cl; pH 7.5
3. 5X Fragmentation buffer (200mM Tris-acetate; pH 8.1, 500mM KOAc, 150mM
MgOAc; see notes)
4. Fragmentation stop solution (0.5M EDTA)
5. Glycogen (20mg/ml)
6. 3M NaOAc
7. Absolute ethanol
8. 70% ethanol (use DEPC water)
9. SuperSCRIPT III (Invitrogen; 200U/µl)
10. 5X First strand buffer (comes with SuperSCRIPT III)
11. DTT (comes with SuperSCRIPT III)
12. Random primers
13. 25mM dNTP mix (alternatively use 10 mM mix)
14. RNAseOUT (Invitrogen; 40U/µl)
15. 10X NE Buffer 2 (Optional: 5X Second strand buffer from Invitrogen; see notes)
16. RNAse H (NEB 5U/µl)
17. E. coli DNA Polymerase I (10U/µl)
18. Bead Binding Buffer
19. Bead Washing Buffer
20. Sera-Mag oligodT beads
Notes on reagent preparation:
It is easy to introduce RNAse in your solutions. All the precautions of working with RNA is
applicable. Always wear gloves while handling these reagents. It is advisable to store them
in a clean dust free place and use for RNA only. Also, keeping smaller aliquots help
avoiding cross contamination.
DEPC treatment:
DEPC is an irreversible RNAse inhibitor which binds very strongly to RNAse and cause
covalent modification of histidine residues. It is effective at 0.1-0.2% concentration. Higher
concentration is needed only when high level of contamination is suspected. But, traces of
DEPC can interfere with downstream enzymatic reactions.
DEPC is highly toxic and volatile. So always use in fume hood. Also, it has a short half life
of 30 min in water and is heat labile. DEPC breaks down into C02, water and ethanol and
smells sweet. Solutions having 0.1% DEPC when autoclaved for 15 mins can safely be
assumed to be DEPC free (http://www.ambion.com/techlib/tb/tb_178.html).
To treat solutions with DEPC, add 1ml/lt of the solution and stir it for at least an hour and
then autoclave for at least 15 mins.
Tris containing solutions CANNOT be treated with DEPC as it reacts with amine groups of
Tris. For this purpose, use RNAse free Tris and dissolve in DEPC treated water in RNAse
free glassware. It can be autoclaved later if you are worried as autoclaving does inactive a
substantial proportion of RNAse.
Stock solutions:
1M Tris-Cl; pH 7.5 (prepare in DEPC water; adjust pH with HCl)
1M Tris-acetate pH 8.1 (prepare in DEPC water; adjust pH with acetic acid)
4M LiCl (treat with DEPC)
3M NaOAc (treat with DEPC)
0.5 EDTA (treat with DEPC)
1M KOAc (treat with DEPC; I purchased 1M RNAse free solution from Sigma)
1M MgOAc (treat with DEPC; I purchased 1M RNAse free solution from Sigma)
Buffer Compositions:
Bead Binding Buffer:
Stock
Final
Volume
Tris-Cl; pH 7.5
1.0M
20mM
1.0ml
LiCl
4.0M
1.0M
12.5ml
EDTA
0.5M
2mM
200µl
DEPC water
37.3ml
TOTAL VOLUME
50.0 ml
Bead Washing Buffer:
Stock
Final
Volume
Tris-Cl; pH 7.5
1.0M
10mM
500µl
LiCl
0.15M
1.0M
1.875ml
EDTA
0.5M
1mM
100µl
DEPC water
47.5 ml
TOTAL VOLUME
50.0 ml
5X Fragmentation Buffer:
Stock
Final
Volume
Tris-acetate; pH 8.1
1.0M
200mM
200 µl
KOAc
1.0M
500mM
500 µl
MgOAc
1.0M
150mM
150µl
DEPC water
150 µl
TOTAL VOLUME
1.0 ml
DAY 1 (mRNA purification and fragmentation)
Step 1: mRNA purification from eukaryotic cells
Consumables:





Sera-mag Magnetic Oligo(dT) Beads
Bead Binding Buffer
Bead Washing Buffer
Ultra Pure Water
10 mM Tris-Cl; pH 7.5
Procedure:
1. Dilute the total RNA with nuclease-free water to 50 μl in a 1.5 ml RNase-free non-sticky
tube. Normal eppendorf tube also works fine.
2. Heat the sample at 65°C for 5 minutes to disrupt the secondary structures and then place
the tube on ice.
3. Aliquot 15 μl of Sera-mag oligo(dT) beads into a 1.5 ml RNase-free non-sticky tube.
4. Wash the beads two times with 100 μl of Bead Binding Buffer and remove the
supernatant.
5. Resuspend the beads in 50 μl of Bead Binding Buffer and add the 50 μl of total RNA
sample from step 2.
6. Aliquot 50 μl of Binding Buffer to a fresh 1.5 ml RNase-free non-sticky Eppendorf tube.
7. Rotate the tube from step 5 at room temperature for 5 minutes and remove the
supernatant. 8. Wash the beads twice with 200 μl of Washing Buffer and remove the
supernatant.
9. Add 50 μl of 10 mM Tris-HCl to the beads and then heat at 80°C for 2 minutes to elute
the mRNA from the beads.
10. Immediately put the tube on the magnet stand, transfer the supernatant (mRNA) to the
tube from step 6, and add 200 μl of Washing Buffer to the beads.
11. Heat the samples at 65°C for 5 minutes to disrupt the secondary structures and then
place the samples on ice.
12. Wash the beads from step 9 twice with 200 μl of Washing Buffer and remove the
supernatant.
13. Add 100 μl of the mRNA sample from step 11 rotate it at room temperature for 5
minutes, and remove the supernatant.
14. Wash the beads twice with 200 μl of Washing Buffer and remove the supernatant.
15. Add 17 μl of 10 mM Tris-HCl to the beads and heat at 80°C for 2 minutes to elute the
mRNA from the beads.
16. Immediately put the tube on the magnet stand and then transfer the supernatant (mRNA)
to a fresh 200 μl thin-wall PCR tube. The resulting amount of mRNA should be
approximately 16 μl.
17. Proceed immediately to RNA fragmentation.
Notes:
It is important that the beads are thoroughly mixed into a homogeneous suspension before
use. Also, never allow the beads to dry as it lowers the yield substantially. Add the next
buffer as soon as you remove one while the tubes are still on magnetic stand. Then vortex
and proceed with next tube.
I used beads that came with the Illumina kit as they supplied a large quantity (I used for ~
25 isolations and still have some left).
Oligo-dT beads can be purchased from many vendors including NEB and Invitrogen and
they provide their recommended buffer compositions as well. I used the buffer composition
provided in older versions of the Illumina protocol for Digital Gene Expression. However,
all are very similar and mine should work with NEB/Invitrogen beads. The only difference
is Invitrogen buffer contains LiDS. This buffer is also used to lyse cells and one can capture
mRNA from the lysates straight away. That is why it has the additional LiDS.
These beads are stable at 4oC and can also be regenerated to isolate RNA from a different
source. Add 0.1 N NaOH to the beads and incubate at room temperature with agitation for 5
minutes. Wash beads twice with sterile RNase-free 1X PBS (pH 7.4) containing 0.1%
Tween 20. Then store in the same buffer.
Step 1: mRNA purification from prokaryotic cells
For prokaryotic cells deplete the rRNA using a commercially available kit such as
MicrobExpress (Ambion), RiboZero (Epicentre) or RiboMinus (Invitrogen). Ensure the
depleted RNA is a volume of 16ul and then proceed with RNA fragmentation.
Step 2: RNA fragmentation
Consumables







5X Fragmentation Buffer
Fragmentation Stop Solution
Glycogen
Ultra Pure Water
3 M NaOAC, pH 5.2
70% EtOH
100% EtOH
Procedure:
1. Prepare the following reaction mix in a 200 μl thin wall PCR tube:
 5X Fragmentation Buffer
 mRNA
4 μl
16 μl
2. Incubate the tube in a PCR thermal cycler at 94°C for exactly 5 minutes. The thermal
cycler should be at 94°C before putting the tube in to avoid ramping time.
3. Add 2 μl of Stop Buffer.
4. Place the tube on ice.
5. Transfer the solution to a 1.5 ml microcentrifuge tube.
6. Add the following to the tube and incubate at 4°C for overnight (see notes)
 3 M NaOAC, pH 5.2
 Glycogen
 100% EtOH
10 μl
2 μl
200 μl
(RNA can be left at -20oC at this stage for longer periods)
Notes:
The RNA fragmentation buffer composition is from Mortazavi et al., 2009. This is also
available from Affymetrix. Another alternative is to use 10X Fragmentation Reagent from
Ambion (which probably is 100mM Tris-Cl; pH7.0, 100mM ZnCl2). I tried this at 70oC for
10, 20 and 30 min and found RNA was fragmented to smaller than 200bp sizes although the
distribution was quite tight. However, the time needs to be optimized and I think 2-5 min
incubation will do the trick.
It is recommended that you process 1-2 samples at a time. Otherwise, incubation time of
later samples will be longer and RNA will be fragmented to smaller sizes.
Low temperature (-20 or -80oC) incubation does not help nucleic acid precipitation. If
anything, this lowers yield due to higher viscosity of ethanol at low temperature. Better
recovery has been observed at times only with overnight incubation at 4oC. However, with
glycogen as carrier protein this may not be needed. However, I have not tested this.
Also, it is the time and speed of centrifugation that affect precipitation most. Hence, the
longer spins.
I am fastidious about RNA/mRNA and prefer to leave them in ethanol rather than in water
at -20 or -80oC. Also, it suits my work flow better. If you are going ahead with mRNA
precipitation as in Illumina protocol, I would suggest proceeding straight to cDNA synthesis
(at least to 1st strand). It can then be safely stored at -20oC.
DAY 2: (1st strand and 2nd strand cDNA synthesis)
Step 3: 1st strand cDNA synthesis
Consumables:






25 mM dNTP Mix
Random Primers
RNaseOUT
100 mM DTT
SuperScript III
5X First Strand Buffer
Procedure :
1. Centrifuge the tube at 14,000 rpm (20,200 relative centrifugal force) for 25 minutes at
4°C in a microcentrifuge
2. Carefully pipette EtOH without dislodging the RNA pellet.
3. Wash the pellet with 300 μl of 70% EtOH.
4. Centrifuge the pellet and carefully pipette out the 70% EtOH.
5. Air dry the pellet for 10 minutes.
6. Resuspend the RNA in 11.1 μl of RNase-free water
7. Assemble the following reaction in a 200 μl thin wall PCR tube
 Random Primers (1 μl)
 mRNA (11.1 μl)
8. Heat the samples at 65°C for 5 min in a PCR machine and put immediately on ice. After
at least two min, add to the sample:




5X First Strand Buffer (4 μl)
100 mM DTT (2 μl)
25 mM dNTP Mix (0.4 μl)
RNaseOUT (0.5 μl)
If processing multiple samples mix the following reagents in the order listed in a separate
tube. Multiply each volume by the number of samples being prepared. Prepare 10% extra
reagent. Add 6.9 μl of mixture to the PCR tube and mix well.
9. Heat the sample at 25°C in a thermal cycler for 2 minutes.
10. Add 1 µl of SuperSCRIPT to the sample and incubate in a thermal cycler as follows:
 25°C for 10 minutes
 42°C for 50 minutes
 70°C for 15 minutes
 Hold at 4oC
11. Place the tube on ice.
Notes:
I used Illumina supplied primers as they supplied a large amount with the kit. It can be
purchased separately from many vendors including NEB. Dissolve it in RNAse free water.
Step 4: 2nd strand cDNA synthesis
Consumables:





Sterile MiliQ water
5X Second Strand Buffer (Invitrogen)
25mM dNTP mix
E. coli DNA polymerase (NEB; 10U/ µl
RNAse H (NEB; 5U/ µl)
Procedure :
1. Keeping the tube on ice add the following to 1st strand cDNA reaction:






Water
25mM dNTP
10X NE Buffer 2
5X Second Strand Buffer
E. coli DNA polymerase
RNAse H
52.8 µl
1.2 µl
20 µl
5 µl
1 µl
62.8 µl
1.2 µl
10.0 µl
5 µl
5 µl
2. Mix well, spin and incubate at 16oC for 2.5 hrs
3. Using QIAquick PCR purification kit purify now double stranded cDNA and elute in 50
µl EB. Pre-warm EB to 50oC to ensure better recovery.
4. Store at -20°C.
N.B. Send fragmented cDNA on dry ice.
Notes:
Invitrogen’s 5X Second Strand Buffer contain NAD+ which is an essential co-factor for E.
coli DNA ligase. However, this is not used in this protocol.
NEB E. coli DNA polymerase acts in NEBuffer 2. NEB RNAseH is also active in NEB
Buffer 1-3. And NEB recommends use of NEBuffer 2 when DNA polymerase and RNAseH
are used together. I used 5XSSB (Invitrogen) for one library and 10X NEB for another.
Both worked fine.