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 ()
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 4 μl
· mRNA 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 10 μl
· Glycogen 2 μl
· 100% EtOH 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 52.8 µl 62.8 µl
· 25mM dNTP 1.2 µl 1.2 µl
· 10X NE Buffer 2 - 10.0 µl
· 5X Second Strand Buffer 20 µl -
· E. coli DNA polymerase 5 µl 5 µl
· RNAse H 1 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.