FGRS: protocol

 

RNA-seq Library Preparation

Last Updated

March 3, 2011 5:13 PM


Modified from Eli Meyer’s protocol:
Preparation of cDNA fragment libraries for SOLiD sequencing


Courtesy of: Iyer Lab, Lauren Fairchild


Note:  Please see the Primers & Process at the bottom of the protocol for a better description of primers.


1.  Total RNA Preparation

2.  RNA Fragmentation


NOTE:  The volume and concentration of the RNA, and also the MgCl2, is critically important for the success of fragmentation. It’s a good idea to test some RNA from the same source as the sample intended for SOLiD sequencing at a range of times to identify the ideal incubation period for that sample.


RNA fragmentation buffer = 100 mM Tris (pH 8.0), 2 mM MgCl2

Note that you should make 1M Tris (pH 8.0) and 100 mM MgCl2 stocks and then make the fragmentation buffer solution as dilutions of these stocks.

  1. a.Master mix 2.25 µg of Total RNA into RNA fragmentation buffer such that the total volume is 90 µl.  Mix this mixture well.

    Note that the 2.25 µg must be accurate - do NOT pipette less than 1.0 µL volume. 

    If your RNA is at a concentration greater than 2.25 ug / µL you will need to make a dilution.  It is important to not dilute your RNA so far, however, that the RNA volume added is greater than 5 µL (the RNA fragmentation buffer volume added should be 85 µL or greater).  This means that the
    maximum RNA concentration for this step is 2.25 ug / µL and the minimum RNA concentration is 450 ng / µL.

  2. b.Aliquot the master mix into 8 separate 10 µl reactions (PCR tubes).

    Reserve the additional sample of total RNA for gel analysis (~10 uL).

  3. c.Incubate the 8 PCR tubes at 95°C for 2, 3, 4, 5, 6, 7, 8 and 9 minutes.

    This is accomplished through careful timing and removal from the thermal cycler at specific time intervals.  Always place the tubes on ice immediately after removal.

    Use thermal cycler program RSEQFRAG.

  4. d.Evaluate the fragmented RNA, along with the un-fragmented sample of RNA, on an agarose gel to confirm the extent of fragmentation.  Note that you should use a low volume of ladder (~3 uL) in order to allow the samples to be more visible.

    See below for example(s).

           

    Gel analysis of 0, 2, 4, 6, 5, 8, 9, and 10 minutes RNA fragmentation.
    Note that you will be performing 0, 2, 3, 4, 5, 6, 7, 8 and 9 minutes.

    This is an optimal result.  Fragmentation begins at 2 minutes and fragments converge to between 500 and 250 bp.  This gel would show us that 6 minutes produces the appropriate amount of fragmentation for this sample.

    Let’s look at a few problematic results.

           

    This RNA is not fragmented at all (across time).  In this case you would make a new fragmentation buffer using an increased concentration of MgCl2.  You should try concentrations of 4 mM, 6 mM, 8 mM, etc until you achieve desired fragmentation.

           

            Another example of incomplete fragmentation.

           

            An example of over fragmentation.  You should try concentrations of 1 mM, 0.5 mM, etc until you achieve desired fragmentation.

    Do not proceed to step (e) until you have determined the time at 95°C and concentration of MgCl2 that is appropriate for your RNA sample.

    Time: _______ minutes   MgCl2 Concentration: _______ mM

  5. e.Master mix 2.25 µg of Total RNA into RNA fragmentation buffer such that the total volume is 90 µl.  Mix this mixture well.

    Note that the concentration of MgCl2 is as determined in step (d).

    Note that the 2.25 µg must be accurate - do NOT pipette less than 1.0 uL volume.

  6. f.Aliquot master mix into 8 separate 10 µl reactions (PCR tubes).

    You will pool these 8 separate reactions back together in step (h) below.

  7. g.Incubate the 8 PCR tubes at 95°C for the time determined in step (d).

    This is accomplished through careful timing and removal from the thermal cycler at a specific time.  Always place the tubes on ice immediately after removal.

    Use thermal cycler program RSEQFRAG.


  1. h.Pool the fragmented RNA aliquots for each sample, and precipitate with sodium acetate and 100% ethanol (at least one hour, possibly overnight).  Dissolve the pellet in 4 µl 10 mM Tris.

    NOTE: At this point - do NOT quantitate your RNA concentration with the NanoDrop.  You will use too much of your product.  Simply proceed.


3.  First-strand cDNA Synthesis


NOTE:  This protocol assumes we started with > 2 µg of total RNA (before fragmentation).  Your pooled sample at step (2h) is likely ~ 1 µg.


  1. a.Combine all of the fragmented RNA from (2h) with 1 µl of 10 µM primer 3SLD-TVS, add H2O to a final volume of 5 µl.

  2. b.Incubate at 65°C for 3 minutes, then transfer onto ice.

  3. c.Prepare a cDNA synthesis master mix containing, for each reaction:

  4. 0.5 µl    H2O

  5. 0.5 µl    dNTPs

  6. 1 µl      DTT (comes with SuperScript III)

  7. 2 µl      5X FS Buffer (comes with SuperScript III)

  8. 0.5 µl    10 µM S-SLD-SW

  9. 0.5 µl    SuperScript III Reverse Transcriptase

  10. d.Add 5 µl of this master mix to the RNA from (3b), mix well, and incubate for one hour at 42°C (heat block).

  11. e.Incubate at 65°C for 5 minutes to inactivate the RT, dilute 1:5 in H2O, and hold on ice.


4.  cDNA Amplification


NOTE:  It is important not to over-amplify the cDNA at this stage.  If a visible smear cannot be produced in 17-19 cycles, try adding more template (diluted FS-cDNA).  The template volume must not exceed 1/10 of the total reaction volume (3 µl), so if this amount of template and cycles still does not produce a visible smear, consider troubleshooting the first-strand synthesis.


  1. a.Prepare a PCR master mix containing the following:

  2. 12 µl       cDNA template from step (3e)

  3. 3 µl        dNTPs

  4. 12 µl       10X PCR buffer (comes with AmpliTaq)

  5. 2.4 µl      AmpliTaq Polymerase

  6. 89.8 µl     H2O


  1. b.Divide this mix into four tubes (A, B, C, D) and add the following to each, per reaction:

    Tube Addition

A    1.2 µl H2O

B    0.6 µl 10 µM 3SLD-TVS, 0.6 µl H2O

C    0.6 µl 10 µM 5SLD, 0.6 µl H2O

D    0.6 µl 3SLD-TVS, 0.6 µl 5SLD

  1. c.Amplify using the following program:

    95°C 5 min, (95°C 40 sec, 63°C 1 min, 72°C 2 min) X 17
    Use thermal cycler program RSEQCAMP.

  2. d.After 17 cycles check 3 µl of the PCR products for all reactions on a gel.  If a visible smear is detected in the appropriate reaction (D) and the controls are clean (A-C), the product is ready for the next stage.  If no product is visible, add 1-2 cycles at a time and check it on a gel until a visible product is formed.  If no product is visible before 20 cycles, consider repeating this step (4) with more cDNA template.

  3. e.Once the appropriate amount of template and number of cycles has been determined, prepare multiple (3 to 6) reactions as described above for mix D.  Amplify them using the optimum template and cycle numbers, and pool these multiple reactions.

  4. f.Purify the pooled PCR products from (3e) using Qiagen’s Qiaquick PCR purification kit, according to the manufacturers’ instructions.  Elute the final sample in 30 µl of 10 mM Tris.


5.  Adaptor Extension and Size Selection


IMPORTANT NOTE: The size distribution of the templates is critical for successful emulsion PCR, so SOLiD sequencing requires careful size selection prior to emPCR.

The directions below outline a simple procedure for selecting fragments ranging from 150-200 bp in size.


  1. a.Quantify the purified products from (4f) and calculate the volume required to use 200 ng of this material as template for the next PCR reaction.  Speed-vac to increase concentration if needed.

  2. b.Prepare a PCR master mix containing the following:

  3. 200 ng     cDNA from (4f)

  4. 3 µl       dNTPs

  5. 12 µl      10X PCR buffer

  6. 2.4 µl     AmpliTaq polymerase

  7. x µl       H2O (total volume at this point = 115.2 µl)

  8. c.Divide this mix into four tubes (A, B, C, D) and add the following to each, per reaction:

Tube Addition

A    1.2 µl H2O

B    0.6 µl 10 µM bar-coded adaptor P2 (- strand), 0.6 µl H2O

C    0.6 µl 10 µM multiplex PCR primer 1, 0.6 µl H2O

D    0.6 µl 10 µM bar-coded adaptor P2, 0.6 µl 10 µM multiplex PCR primer 1

  1. d.Amplify using the following program:

    95°C 5 min, (95°C 40 sec, 63°C 1 min, 72°C 2 min) X 4
    Use thermal cycler program RSEQAEXT.

  2. e.After 4 cycles check 3 µl of the PCR products for all reactions on a gel.  If a visible smear is detected in the appropriate reaction (D) and the controls are clean (A-C), the product is ready for the next stage.  If no product is visible, add 1 cycle at a time and check it on a gel until a visible product is formed.  If no product is visible before 6 cycles, consider repeating this step (5) with more template cDNA.

  3. f.When an acceptable product has been achieved, prepare a new gel (or eGel) and load the remainder of each reaction into a single well.  After running the gel, cut out a band from the smear that corresponds to 150-200 bp in size.

    A low molecular weight ladder is useful for evaluating these small size differences.  Exclude the edges of the lane from the gel slice, and minimize the total mass of this slice - trim away the parts of the slice that don’t contain much DNA.

  4. g.Transfer gel slice into a PCR tube.

  5. h.Extract the cDNA fragments using agarose gel extraction kit.


6.  Amplification of Size-selected Fragments (Diagnostic Test)


IMPORTANT NOTE: This step is a useful test of the final constructs, but is not required for SOLiD sequencing, which only requires about 0.5-1.0 pg of input cDNA.  This is a diagnostic test only - your final product for sequencing was produced in step (5h).


  1. a.Approximately 1-5 ng of cDNA will be enough template for this PCR.  This is typically 1-3 µl of product from gel extraction (5h).

  2. b.Prepare a PCR master mix containing the following:

  3. 8 µl        cDNA from step (5h)

  4. 3 µl        dNTPs

  5. 12 µl       10X PCR buffer

  6. 2.4 µl      AmpliTaq polymerase

  7. 89.8 µl     H2O

  8. c.Divide this mix into four tubes (A, B, C, D) and add the following to each, per reaction:

    Tube Addition

A    1.2 µl H2O

B    0.6 µl 10 µM Lib PCR 1, 0.6 µl H2O

C    0.6 µl 10 µM Lib PCR 2, 0.6 µl H2O

D    0.6 µl Lib PCR 1, 0.6 µl Lib PCR 2

  1. d.Set up four reactions per template: one with each of the mixes A-D.

  2. e.Amplify using the following program:

    95°C 5 min, (95°C 30 sec, 63°C 30 sec, 72°C 30 sec) X 8
    Use thermal cycler program RSEQFAMP.

  3. f.After 8 cycles check 3 µl of the PCR products for all reactions on a gel.  If a clean, single band at 150-200 bp is detected in the appropriate reaction (D) and the controls are clean (A-C), the constructs are correct.  If no product is visible, add 2 cycles at a time and check it on a gel until a visible product is formed.  If no product is visible before 12 cycles, consider repeating this step (6) with more template cDNA.

  4. g.Once the constructs have passed the PCR tests in (6f) they are ready for SOLiD sequencing.  The purified template from step (5h) is the material to be sequenced.



Primers & Process Reference (provided by Lauren Fairchild)

The slides below explain the process of RNA-seq library preparation in more detail.  The first step is First-strand cDNA Synthesis as shown below.


The primer 3SLD-TVS drives this process.  Note that the knowledge that reverse transcriptase adds terminal Cs as it dissociates from template RNA is important.  These terminal Cs provide homology to the S-SLD-SW primer.



The complementary strand of cDNA is primed by S-SLD-SW.  Double-stranded cDNA results.



Double-stranded cDNA is PCR-amplified with the primers 3SLD-TVS and 5SLD (during the cDNA Amplification step).



Sequencing primers bar-coded adaptor P2 and multiplex PCR primer 1 are attached during the process of Adaptor Extension and Size Selection. This is followed by Amplification of Size-selected Fragments - diagnostic test of your final product.




Primers & Process Reference (Older)


This information is similar to the references provided above.