Directional-RNAseq Prep: Difference between revisions

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The modifications we adapted are those from Parkhomchuk et al. (Nucl. Acids Res., 2009,  
To make strand-specific mRNA libraries, we adapted the method of Parkhomchuk et al. (Nucl. Acids Res., 2009,  
doi: 10.1093/nar/gkp596). This was determined by Levin et al. (Nat Methods 2010, 7:709-715) to be one of the most reliable strand-specific sequencing method tested. While our method is optimized for the Illumina TruSeq RNA sample preparation kit, it can likely be used for any library construction method that procedes via polymerase-mediated double-stranded cDNA/second strand synthesis.
doi: 10.1093/nar/gkp596). In a comparison of several different strand-specific methods, Levin et al. (Nat Methods 2010, 7:709-715) found the dUTP method to be among the most reliable tested. Our method has been optimized for the Illumina TruSeq RNA sample preparation kit, but it can likely be used for any library construction method that procedes via polymerase-mediated double-stranded cDNA/second strand synthesis.


==Materials required for making Strand-Specific mRNAseq Illumina Libraries via dUTP==
==Materials required for making Strand-Specific mRNAseq Illumina libraries via dUTP==


• 48 well plate or RNase-free, non-sticky 1.5 mL tubes
• 48 well plate or RNase-free, non-sticky 1.5 mL tubes


• Illumina TruSeq RNA Sample preparation kit(Illumina #RS930-2001; manual 15008136 Rev. A, November 2010)
• Illumina TruSeq RNA Sample preparation kit (Illumina #RS930-2001; manual 15008136 Rev. A, November 2010)


• Superscript III first strand synthesis kit (Invitrogen 18080-051)
• Superscript III first strand synthesis kit (Invitrogen 18080-051)
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• NEBNext Second Strand Synthesis Buffer, dNTP-Free (NEB B6117S)
• NEBNext Second Strand Synthesis Buffer, dNTP-Free (NEB B6117S)


• NEBNext mRNA Second Strand Synthesis Module (NEB E6111S)
• NEBNext mRNA Second Strand Synthesis enzyme mix (NEB E6111S)


• 2 mM dNTP/ 4 mM dUTP Mix (Fermentas R0251)
• 2 mM dNTP/ 4 mM dUTP Mix (Fermentas R0251)
Line 19: Line 19:


• 5M NH4OAc; Glycoblue (Ambion) or similar coprecipitant; 100% Ethanol; RNAse-free water
• 5M NH4OAc; Glycoblue (Ambion) or similar coprecipitant; 100% Ethanol; RNAse-free water


==These steps replace or are added to the Illumina TruSeq RNA Sample preparation guide==
==These steps replace or are added to the Illumina TruSeq RNA Sample preparation guide==
Line 27: Line 26:
1. Perform First Strand Synthesis (FSS) reaction as directed.  
1. Perform First Strand Synthesis (FSS) reaction as directed.  
   
   
2. When the FSS reaction is complete, precipitate RNA with ethanol and ammonium acetate.  Per 50 ul FSS reaction, mix:


    • 5 ul 5M NH4OAc (ammonium is used because it does not precipitate nucleotides).
'''''---- When the FSS reaction is complete, clean RNA using Sephadex G-50 ---'''
    • 2 ul GlycoBlue (or similar co-precipitant)
 
    • 110 ul 100% Ethanol
2.  Add dry Sephadex G-50 (Sigma, cat# G-50-150) to Millipore 45μL column loader (Millipore, cat# MACL 09645).
 
3.  Remove excess resin from the top of the column loader with the supplied scraper.
 
4.  Place the multiscreen HV plate (Millipore, cat# MAHV N45) upside-down on top of the column loader and invert both multiscreen HV plate and column loader.
 
5.  Tap on the top of the column loader to release the resin.
 
6.  Add 300 μL of dH<sub>2</sub>O to each well. Let stand at room temperature for 3 hours to allow resin to swell.
 
7.  Once resin has swollen in the Multiscreen plates, these plates can be sealed with Saran Wrap and stored at 4°C for several weeks in a sealed plastic container containing a damp paper towel to assure they are kept moist.
 
8.  To prepare for use, place the G50-containing Multiscreen filter plate on top of a 96-well receiver. Stabilize this "sandwich" with 2 pieces of lab tape.
 
9.  Centrifuge the sandwich for 5 minutes at 2500 rpm (1000 x g) at room temperature. This packs the columns and removes excess water.
 
10. Undo sandwich and remove the water in the 96-well receiver. Re-make sandwich and add 150 μL dH<sub>2</sub>O to each well in the G50-containing Multiscreen filter plate and spin for an additional 5 min at 2500 rpm at room temperature.


3. incubate 1 hour to overnight at -80°C
11. Disassemble the sandwich and discard the water that collected in the 96-well receiver.


4. centrifuge 30 min x 20,000g, 4°C
12. Add samples to the G50-containing Multiscreen filter plate, making sure to not disturb the resin.(ideal minimum volume is 10μL; max volume is 100μL).


5. Remove supernatant; add 200 ul of ice-cold 70% EtOH
13. Place the G50-containing Multiscreen filter plate on top of a new 96-well receiver plate. Stabilize this "sandwich" with 2 pieces of lab tape.


6. centrifuge 5 min x 20,000g, 4°C
14. Spin at 2500 rpm for 5 min at room temperature; spin again if filtered volume is less that 20μL.


7. Remove supernatant; air dry 10 minutes.
15. Transfer samples to a new 96-well pcr plate. Samples are now ready for the next step.




'''''---- Resuspend pellet in FSS buffer w/o dNTPs or RT (new step) ---'''''
'''''---- To Sephadex cleaned FSS products (new step) ---'''


1. Per sample, mix the following reagents from the Superscript III kit together in a master mix:
1. Per sample, mix the following reagents from the Superscript III kit together in a master mix:


     • 2 ul 10X RT buffer  
     • 1 ul 10X Reverse Transcription buffer  
     • 4 ul 25 mM MgCl2
     • 2 ul 25 mM MgCl2
     • 2 ul 0.1 M DTT
     • 1 ul 0.1 M DTT
     • 1 ul random hexamers (50 ng/ul)
     • 0.5 ul random hexamers (50 ng/ul)
    • 49 ul dH2O


2. Add 58 ul of the master mix to each pellet and resuspend by pipetting gently.  
2. Add 4.5 ul of the master mix to each well and mix by pipetting gently.  




Line 60: Line 73:


1. Per sample, mix the following into a master mix:
1. Per sample, mix the following into a master mix:
     • 8 ul of 10X dNTP-FREE Second Strand buffer (NEB B6117S)
     • 4 ul of 10X dNTP-FREE Second Strand buffer (NEB B6117S)
     • 10 ul dNTP/dUTP mixt
     • 5 ul dNTP/dUTP mixture
     • 4 ul Second Strand enzyme mix (NEB E6111S)
     • 2 ul Second Strand enzyme mix (NEB E6111S)


2. Pipette 22 ul of the master mix into each resuspended RNA to give a final volume 80 ul.
2. Pipette 11 ul of the master mix into each resuspended RNA to give a final volume 40 ul.


3. Incubate 2.5 hours at 16C.
3. Incubate 2 hours at 16°C.




Line 79: Line 92:
1. Per sample, add 1 ul of USER enzyme mixture.
1. Per sample, add 1 ul of USER enzyme mixture.


2. Incubate at 37C for 15 minutes, then place on ice  
2. Incubate at 37°C for 15 minutes, then place on ice  




Latest revision as of 10:58, 5 April 2012

To make strand-specific mRNA libraries, we adapted the method of Parkhomchuk et al. (Nucl. Acids Res., 2009, doi: 10.1093/nar/gkp596). In a comparison of several different strand-specific methods, Levin et al. (Nat Methods 2010, 7:709-715) found the dUTP method to be among the most reliable tested. Our method has been optimized for the Illumina TruSeq RNA sample preparation kit, but it can likely be used for any library construction method that procedes via polymerase-mediated double-stranded cDNA/second strand synthesis.

Materials required for making Strand-Specific mRNAseq Illumina libraries via dUTP

• 48 well plate or RNase-free, non-sticky 1.5 mL tubes

• Illumina TruSeq RNA Sample preparation kit (Illumina #RS930-2001; manual 15008136 Rev. A, November 2010)

• Superscript III first strand synthesis kit (Invitrogen 18080-051)

• NEBNext Second Strand Synthesis Buffer, dNTP-Free (NEB B6117S)

• NEBNext mRNA Second Strand Synthesis enzyme mix (NEB E6111S)

• 2 mM dNTP/ 4 mM dUTP Mix (Fermentas R0251)

• USER (uracil-specific excision reagent) Enzyme mix (NEB M5505S)

• 5M NH4OAc; Glycoblue (Ambion) or similar coprecipitant; 100% Ethanol; RNAse-free water

These steps replace or are added to the Illumina TruSeq RNA Sample preparation guide

---- First Strand Synthesis (page 44) ---

1. Perform First Strand Synthesis (FSS) reaction as directed.


---- When the FSS reaction is complete, clean RNA using Sephadex G-50 ---

2. Add dry Sephadex G-50 (Sigma, cat# G-50-150) to Millipore 45μL column loader (Millipore, cat# MACL 09645).

3. Remove excess resin from the top of the column loader with the supplied scraper.

4. Place the multiscreen HV plate (Millipore, cat# MAHV N45) upside-down on top of the column loader and invert both multiscreen HV plate and column loader.

5. Tap on the top of the column loader to release the resin.

6. Add 300 μL of dH2O to each well. Let stand at room temperature for 3 hours to allow resin to swell.

7. Once resin has swollen in the Multiscreen plates, these plates can be sealed with Saran Wrap and stored at 4°C for several weeks in a sealed plastic container containing a damp paper towel to assure they are kept moist.

8. To prepare for use, place the G50-containing Multiscreen filter plate on top of a 96-well receiver. Stabilize this "sandwich" with 2 pieces of lab tape.

9. Centrifuge the sandwich for 5 minutes at 2500 rpm (1000 x g) at room temperature. This packs the columns and removes excess water.

10. Undo sandwich and remove the water in the 96-well receiver. Re-make sandwich and add 150 μL dH2O to each well in the G50-containing Multiscreen filter plate and spin for an additional 5 min at 2500 rpm at room temperature.

11. Disassemble the sandwich and discard the water that collected in the 96-well receiver.

12. Add samples to the G50-containing Multiscreen filter plate, making sure to not disturb the resin.(ideal minimum volume is 10μL; max volume is 100μL).

13. Place the G50-containing Multiscreen filter plate on top of a new 96-well receiver plate. Stabilize this "sandwich" with 2 pieces of lab tape.

14. Spin at 2500 rpm for 5 min at room temperature; spin again if filtered volume is less that 20μL.

15. Transfer samples to a new 96-well pcr plate. Samples are now ready for the next step.


---- To Sephadex cleaned FSS products (new step) ---

1. Per sample, mix the following reagents from the Superscript III kit together in a master mix: 

    •	1 ul 10X Reverse Transcription buffer 
    •	2 ul 25 mM MgCl2
    •	1 ul 0.1 M DTT
    •	0.5 ul random hexamers (50 ng/ul)

2. Add 4.5 ul of the master mix to each well and mix by pipetting gently.


---- Perform Second Strand Synthesis (SSS) with dUTP/dNTP mix (new step) ---

1. Per sample, mix the following into a master mix: 

    •	4 ul of 10X dNTP-FREE Second Strand buffer (NEB B6117S)
    •	5 ul dNTP/dUTP mixture
    •	2 ul Second Strand enzyme mix (NEB E6111S)

2. Pipette 11 ul of the master mix into each resuspended RNA to give a final volume 40 ul.

3. Incubate 2 hours at 16°C.


---- Clean the SSS product with AMPure, as directed (page 48) ---


---- Continue protocol as directed through the AMPure cleaning step following adapter ligation (page 48) ---


---- Prior to PCR enrichment, degrade the dUTP in the coding strand (new step) ---

1. Per sample, add 1 ul of USER enzyme mixture.

2. Incubate at 37°C for 15 minutes, then place on ice


---- Continue to PCR enrichment step (page 62) ---



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