Hewson Lab at Cornell


Team Aquatic Virus - Microbial Oceanography, P.I. Ian Hewson, Ph.D.

Hewson Lab Metatranscriptomics Protocols

in vitro transcription – Ambion MessageAmp II - Bacteria

Time: On and off for 21 hours.
Essentially 6 hours straight followed by 14 h incubation, followed by ½ hr processing

Parallel Compatibility: ~ 8 samples

Reagents needed:
Ambion MessageAmp II - Bacteria Kit
Ice in ice bucket

Preparation:
Ensure wash buffer contains EtOH
Heat block must be at 70oC
Water bath must be at 37oC
Ensure hybridization oven or other water bath is at 42o
Ensure that thermal cycler is free for 2 hr incubation
Water bath at 50oC
Place RNAse free water in 50oC bath

  1. Thaw samples on ice, if frozen, and transfer 5 μl into RNAse-free 1.5 ml microcentrifuge tube.
  2. Polyadenylation step: Incubate samples at 70oC for 10 min, then place on ice for 3 min.
  3. Make a master mix of the following reagents in an RNAse free 1.5 ml microcentrifuge tube, multiplying each reagent by the number of samples + 1 to account for pipetting error.
  4. Nuclease free water                            1.5μl
    10X Poly(A) Tailing Buffer                1.0μl
    RNAse Inhibitor                                 1.0μl
    Poly(A) Tailing ATP                           0.5μl
    PAP                                                     1.0μl

    Add 5 μl of the master mix to each tube, gently mix by flicking, and then quickly spin on benchtop rotor to collect in the bottom of the tube

  5. Incubate the tubes at 37oC for 15 minutes. After incubation, place the tubes on ice.
  6. First strand synthesis: make the following master mix of reagents in an RNAse-free 1.5 ml microcentrifuge tube, multiplying each reagent by the number of samples+1 to account for pipetting error.
  7. Nuclease-free water                3 μl
    T7 Oligo(dT) VN                    1 μl
    10X First Strand Buffer         1 μl
    dNTP Mix                               4 μl
    ArrayScript                             1 μl

    Add 10 μl of the master mix to each tube and mix by inversion.

  8. Place the mixture in a 42oC Hybridization oven or water bath and incubate for 2 hr.
  9. Second strand synthesis: after incubation place the tubes on ice. Transfer the mixtures to PCR tubes in a qPCR rack in an ice bucket with ice. Make the following master mix of reagents on ice in an RNAse-free 1.5 ml microcentrifuge tube, multiplying each reagent by the number of samples+1 to account for pipetting error.
  10. Nuclease free water                63 μl
    10X Second Strand Buffer     10 μl
    dNTP Mix                               4 μl
    DNA Polymerase                    2 μl
    RNAse H                                1 μl

    Add 80 μl of the master mix to each PCR tube, mix by inversion, and place back on ice. Take to the thermal cycler

  11. Program the thermal cycler as follows: 61 minutes at 16oC, 60 minutes at 16oC, then 4oC for infinity (1 cycle). Hit “Start Run” with 100 μl reaction volume without the tubes in the machine, and open lid. Allow to run for ~ 1 minute.
  12. Place the PCR tubes in the thermal cycler and leave the lid open. It is essential that the tubes do not exceed 16oC during this process. Incubate for 2 h.
  13. After 2 h incubation, place the tubes on ice. Transfer the reactions to RNAse-free 1.5 ml tubes.
  14. Add 250 μl cDNA binding buffer to each tube and invert several times to mix.
  15. Place a cDNA purification column in a catch tube (kit supplied) and then pipette the mixture into the column. Spin at 10,000 x g for 30 s. Discard the flow through and replace in catch tube.
  16. Add 500 μl wash buffer to each column and the centrifuge at 10,000 x g for 60 s. Place the column in a fresh RNAse-free 1.5 ml microcentrifuge tube.
  17. Add 20 μl Nuclease-free water (at 50oC) to the center of the column matrix and allow to stand for 2 min. After this time, centrifuge at 10,000 x g for 60 s. Discard the column, close the catch tube and proceed to the next step.
  18. In vitro transcription: make the following master mix of reagents on ice in an RNAse-free 1.5 ml microcentrifuge tube, multiplying each reagent by the number of samples+1 to account for pipetting error.
  19. T7 ATP                                   4 μl
    T7 CTP                                    4 μl
    T7 GTP                                   4 μl
    T7 UTP                                   4 μl
    10X T7 Reaction Buffer         4 μl
    T7 Enzyme Mix                      4 μl

    Add 24 μl of the master mix to each sample tube (containing around 16 μl – 4 μl gets lost in the elution) and mix gently by inversion.

  20. Incubate the mixtures for 14 h in the 37oC oven. This is normally best done by starting at 7 pm and terminating at 9 am.
  21. aRNA  Purification: at the conclusion of the IVT step, add 60 μl nuclease-free water. Place an aRNA column in a catch tube for each sample being processed.
  22. Add 350 μl aRNA Binding buffer and mix by vortexing.
  23. Add 250 μl 100% EtOH to each sample and mix immediately by inversion (don’t vortex). Immediately pipette the mixture into the aRNA column and spin at 10,000 x g for 30 s. Discard the flow-through.
  24. Add 650 μl Wash Buffer to the columns and centrifuge at 10,000 x g for 30 s. Discard the flow-through. Centrifuge again for 1 min at 10,000 x g and place the aRNA column in a fresh RNAse-free 1.5 ml microcentrifuge tube.
  25. Add 75 μl of Nuclease-free water (at 50oC) to the centre of the column matrix and allow to stand for 2 min, then spin at 10,000 x g for 60 s. Repeat for another 75 μl. After second centrifugation, discard the column and label the tube with sample number and “aRNA”.
  26. Quantify the aRNA using the Bioanalyzer (fine for aRNA since no macromolecules present). Should be at this stage tens of μg of RNA in total volume.  The aRNA can be now stored at -80oC indefinitely.

QA/QC: Quantify the aRNA using the Nanodrop. If < 100 ng/μl clean and concentrate using the Zymo Research RNA Cleanup Kit -5, then re-amplify the sample using the aRNA as template. For eukaryotes follow the protocol for “second round amplification”. For prokaryotes follow the same procedure as the first round.

If samples >100 ng/μl, concentrate them in speed vac to 30 μl. Re-quantify on nanodrop. Make 3 x dilutions of each sample to 454 ng/μl (11 μl total) in PCR tubes.


Conversion of aRNA to double stranded cDNA

Parallel Compatibility: > 20 samples (run 3 replicates for each sample)

Reagents needed:
Invitrogen 5 X First Strand Buffer
Invitrogen dNTP mix
Invitrogen dithiothreitol (DTT)
Invitrogen SuperScript III
Promega Random Hexamers HC
Invitrogen  5 X Second Strand Buffer
Invitrogen E. coli DNA Ligase
Invitrogen E. coli DNA Polymerase I
Invitrogen RNAse H
Invitrogen T4 DNA polymerase
RNAse A (provided in plasmid kits; dilute to 10 mg ml-1
Phenol:Chloroform:Isoamyl Alcohol (24:1:0.1)
NH4OAc (7.5M)

Preparation:
Heat block must be at 70oC
A water bath must be at 50oC
A water bath at 37oC
Make sure PCR Machine is free for 2 h incubation
Place a 15 ml tube of 100 % EtOH in the -80oC freezer
Place a 15 ml tube of 70% EtOH in the -20oC freezer

  1. First Strand Synthesis: thaw aRNA sample dilutions if necessary. [Pipette 5 μg aRNA into a fresh RNAse-free PCR tube. Add Nuclease-free water to a total of 11 μl].
  2. To the aRNA sample add 1 μl Promega Random Hexamers HC (500 ng μl-1) and 1 μl dNTP Mix.
  3. Heat aRNA mixture to 70oC for 10 minutes in a thermocycler. After incubation, place the reactions on ice / frozen rack, and centrifuge briefly to collect at the bottom of the tube.
  4. Make the following master mix of reagents on ice in an RNAse-free 1.5 ml microcentrifuge tube, multiplying each reagent by the number of samples+1 to account for pipetting error.
  5. 5X First Strand Buffer           4 μl
    DTT (0.1 M)                            1 μl
    Superscript III                         2 μl

    Add 7 μl to each reaction tube.

  6. Incubate first strand reactions at 50oC for 60 min in a thermal cycler. After incubation, place on ice or in frozen rack.
  7. Second strand synthesis: make the following master mix of reagents on ice in an RNAse-free 1.5 ml microcentrifuge tube, multiplying each reagent by the number of samples+1 to account for pipetting error. It is imperitive that the mix remain on ice.
  8. Nuclease-free water                88 μl
    5 X Second Strand Buffer      30 μl
    dNTP Mix                               6 μl
    E. coli DNA Ligase                1 μl
    E. coli DNA Polymerase I      4 μl
    RNAse H                                1 μl

    Add 130 μl of master mix to each PCR tube on ice or frozen rack and take to PCR Machine.

  9. Program the thermal cycler as follows: 61 minutes at 16oC, 60 minutes at 16oC, then 4oC for infinity (1 cycle). Hit “Start Run” with 100 μl reaction volume without the tubes in the machine, and open lid. Allow to run for ~ 1 minute.
  10. Place the PCR tubes in the thermal cycler and leave the lid open. It is essential that the tubes do not exceed 16oC during this process. Incubate for 2 h.
  11. After 2 h incubation, add  2 μl T4 DNA polymerase to each tube. Incubate at 10 min at 14oC. After final incubation, place on ice.
  12. RNAse A cleanup: add 1 μl of RNAse A to each tube, mix by inversion and incubate at 37oC for 20 minutes.
  13. After incubation,  add 153 μl of Phenol:Chloroform:Isoamyl Alcohol (24:1:0.1), to fresh RNAse-free 1.5 ml microcentrifuge tubes, and add the samples to the tubes. Invert ten times to mix.
  14. Centrifuge tubes at 10,000 x g for 2 min at room temperature. Remove the upper layer to a fresh RNAse-free 1.5 ml microcentrifuge tube. Discard the lower layer in the Phenol waste.
  15. ds cDNA Precipitation: to each tube add 16 μl NH4OAc, 7 μl Glycogen (5 mg/ml) and 326 μl 100% EtOH from the -80oC. Mix by inversion. Incubate overnight at -20oC.
  16. Centrifuge tubes at 4oC and at 15,000 x g for 30 min. After spinning, remove supernatant with P-1000 pipette, being careful to avoid the pellet.
  17. Add 500 μl 80% EtOH from the -20oC. Centriguge tubes at 15,000 x g at 4oC for 5 min.  Remove the supernatant avoiding the pellet using a P-1000 pipette.
  18. Place open tubes in a speed vac at “medium setting” for 15 minutes to dry the pellet. Be careful to turn the rotor on before releasing the vacuum!! You can lose all your work here!!!
  19. Add 20 μl Nuclease-free water to the dried pellets and resuspend by vortexing. Leave to resuspend at room temperature for 20 minutes. Combine replicate samples.
  20. Measure cDNA quantity on the Nanodrop. ds cDNA samples can be stored at <-20oC indefinitely.  Bear in mind this measurement won’t give you the REAL quantity of ds cDNA in the sample; proceed to next part of the protocol before sending into Schuster Lab/454.

ds cDNA clean-up for Pyrosequencing

Parallel Compatibility: > 20 samples (run 3 replicates for each sample)

Reagents needed:
Zymo Research DNA Clean and Concentrator – 5
Nuclease Free Water
1.5 ml Microcentrifuge tubes
Benchtop microcentrifuge

Preparation:
Ensure the wash buffer has had EtOH added to it

  1. Ds cDNA Cleanup: pipette 40 μl of each ds cDNA sample into 1.5 ml tube (labeled). Add 80 μl of DNA Binding Buffer and mix by vortexing. Briefly centrifuge to collect the mixture to the bottom of the tube
  2. Pipette the mixture into a Spin column in a catch tube. Centrifuge at 10,000 x g for 1 min. Discard the flow-through
  3. Add 200 μl of the wash buffer to the column and spin at 10,000 x g for 1 min. Discard the flow through. Add another 200 μl of wash buffer to the column and spin again at 10,000 x g for 1 min.
  4. Remove the column from the old catch tube and place in a nuclease-free 1.5 ml microcentrifuge tube.
  5. To the column add 20 μl of nuclease free water. Allow to stand for 2 min, then centrifuge at 8,000 x g for 1 min. Add another 20 μl of nuclease free water to the column and centrifuge again for 1 min. Discard the column

QA/QC: Quantify the sample on the nanodrop. It is reasonable to expect the quantity of material in the sample to drop by an order of magnitude from the uncleaned ds cDNA. Run 1 μl of the sample on the bioanalyzer. If there is detectable DNA in the range from 100 – 1000 bp, then send the samples for sequencing. If not, bad luck! Start over!

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