cDNA Synthesis and Amplification Protocol using the Template Switching RT Enzyme Mix (NEB #M0466)

The cDNA synthesis and amplification protocol contains two steps. In the first step, cDNA is synthesized using an RT primer that contains an adaptor of known sequence at the 5′ end. This primer, in conjunction with a template switching oligo (TSO), generates cDNAs containing adaptor sequences at both the 5′ and 3′ ends. In the second step, the resulting cDNAs are directly amplified using primers for the adaptor sequences at the cDNA ends.

Sample Recommendations:

This protocol is optimized for input amounts ranging from 2 pg – 200 ng of intact human total RNA. Cell lysate free of denaturing detergent may also be used as a starting material. In general, the input sample should be free of salts (e.g., Mg2+ and guanidinium), divalent cation chelating agents (e.g., EDTA, EGTA and citrate) or organics (e.g., phenol and ethanol). If an excess amount of genomic DNA is present in RNA samples, an optional DNase I treatment can be performed. Inactivate or remove DNase I after treatment.

For notes and recommendations on other elements of this protocol, please refer to General Guidelines in the last section.

Reaction Preparation

  1. Briefly centrifuge the Template Switching RT Enzyme Mix to collect the solution to the bottom of the tube, then place on ice.
  2. Thaw the Template Switching RT Buffer at room temperature completely. Mix thoroughly by a gentle vortex and centrifuge briefly to collect the solution to the bottom of the tube, then place on ice.

cDNA Synthesis and Template Switching

Please note that the volume needed for each reagent is based on a final cDNA Synthesis and Template Switching reaction volume of 10 μl. The reaction can be scaled up proportionally, while observing the RNA input amount limit, to a final volume of 20 μl.

1.Primer Annealing for 1st Strand Synthesis

1.1 To anneal the RT primer with the RNA templates, in a 0.2 ml nuclease free PCR tube, prepare the reaction as follows (on ice):

Reagent

Volume

Final Concentration

RNA/Lysed Cell

Variable

2 pg -200 ng

RT primer (10 μM)

1 μl

1 μM

dNTP (10 mM)

1 μl

1mM

Nuclease-free Water

Variable

-

Total Volume

6 μl

-

Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube.

1.2 Incubate for 5 minutes at 70°C in a thermocycler with the lid temperature set at ≥ 85°C, then hold at 4°C until next step.

2.Reverse Transcription (RT) and Template Switching

2.1 During the primer annealing reaction, vortex the Template Switching RT Buffer briefly followed by a quick spin to collect the solution to the bottom of the tube, then prepare the RT reaction mix as follows (adding RT Enzyme Mix last):

Reagent

Volume

Final Concentration

Template Switching RT Buffer

2.5 μl

1 X

Template Switching Oligo (75 μM)

0.5 μl

3.75 μM

Template Switching RT Enzyme Mix

1 μl

1 X

Total Volume

4 μl

-

Mix thoroughly by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube.

2.2 Combine 4 µl RT reaction mix (above) with 6 µl of the annealed mix from step 1.2 , mix well by gently pipetting up and down at least 10 times, then centrifuge briefly to collect the solution to the bottom of the tube.

2.3 Incubate the 10 µl combined reaction in a thermocycler with the following steps:

90 minutes at 42°C
5 minutes at 85°C
Hold at 4°C

If not proceeding to the cDNA Amplification by PCR step immediately, samples can be stored at 4°C overnight.

cDNA Amplification by PCR

1. Assemble the cDNA Amplification reaction on ice by adding the following components into the cDNA product from step 2.3 above:

Components

Volume

Final Concentration

Template switching cDNA product (from step 2.3)

10 μl

-

NEBNext High-Fidelity 2X PCR Master Mix (NEB #M0541)*

25 μl

1x

cDNA PCR Primer (10 μM)

1 μl

0.2 μM

H2O

14 μl

-

Total Volume

50 μl

-

*Alternatively, Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494) can be used.

Mix gently and then centrifuge briefly to collect the solution to the bottom of the tube.

2.Incubate in a thermocycler, with lid temperature set at ≥100°C, and perform PCR with the following cycling condition:

Step

Temperature

Time

Cycles

Initial Denaturation

98°C

45 sec

1

Denaturation

98°C

10 sec

Please refer to the table below for cycle recommendation

Annealing

62-68°C*

15 sec

Extension

72°C

3 min

Final Extension

72°C

5 min

1

Hold

4°C

 

*For annealing temperature calculations, please visit http://tmcalculator.neb.com

PCR cycle number recommendations based on Universal Human Reference (UHR) RNA :

Total RNA Input

Number of PCR Cycles*

2 pg

20-21

10 pg

17-18

100 pg

14-15

1 ng

11-12

10 ng

8-9

100-200 ng

7-8

*When using other sources of starting material, some optimization may be necessary due to variations in mRNA amounts.Using the above PCR cycles recommendations, the expected cDNA yields are 5-15 ng for 2 pg -10ng of UHR total RNA and 50-200 ng for UHR total RNA ≥100 ng.

3.Clean up the amplified cDNA library with AMPure or SPRIselect beads (Beckman Coulter) following the manufacturer’s recommendations.

Amplified cDNA may be stored at 4°C overnight or at -20°C for up to a week before or after the clean up step above.

 

General Guidelines

1. RNA template

This protocol is optimized for 2 pg – 200 ng of intact human total RNA using poly(dT) (containing an adaptor of known sequence at its 5´ end) as an RT primer. The template switching efficiency is the highest for RNA template with a cap structure or 5´PPP, therefore, the RNA input amounts and PCR cycle numbers need to be optimized when the input RNA is fragmented.

Cell lysates from 1-100 cells may also used as input; however, the cell lysis buffer should not contain guanidium salts or denaturing detergents (e.g., SDS).

When using a poly(dT) primer containing an adaptor at its 5´ end, rRNA depletion or mRNA isolation is not necessary for eukaryotic total RNA input.

2. RT primer

Depending on the starting material, either random hexamers or poly (dT) can be used at the RT primer. An adaptor of known sequence at the 5´ end of the RT primer is required for either type of RT primer to enable downstream cDNA amplification by PCR.

For poly(dT) primer, an oligo (dT)30 VN is recommended for optimal cDNA yield and specificity.

The best results are typically seen when using the RT primer at a final concentration of 1 μM for total RNA inputs of 2 pg - 5 ng and 2 μM for RNA inputs higher than 5 ng.

3. Template switching oligo (TSO)

Different types of TSO can be used including TSOs with 5´ modifications (e.g., biotin, isomeric bases or abasic spacers). When the starting material is extremely low (≤10 pg of total RNA), we recommend using the following TSO sequence , GCT AAT CAT TGC AAG CAG TGG TAT CAA CGC AGA GTA CAT rGrGrG, or a TSO with 5´ modifications (e.g. biotin, isomeric or abasic bases) to avoid concatemers that may form at a low frequency [1, 2].

A TSO sequence with rGrGrG at the 3´ end is recommended for efficient template switching. For direct cDNA PCR amplification, the presence of an rU containing TSO may cause inhibition if an archeal family B (e.g., high fidelity) DNA polymerase is used.

A final TSO concentration of 1-3.75 μM can be used. For optimal cDNA yield, a final TSO concentration of 3.75 μM is recommended.

4. cDNA amplification

The RT reaction does not need to be purified before cDNA amplification. If a cleanup step is desired, we recommend AMPure XP or SPRIselect beads. The bead to sample volume ratio is dependent on the expected size of the cDNA product. Please refer to the manufactuer’s recommendations for the detailed sample clean up procedure.

If an ExoI treatment is to be applied, Exo I can be directly added after the RT reaction is completed and cooled to 4°C in the thermocycler. A titration of Exo I is recommended and the enzyme must be inactivated prior to the PCR setup step.

5. DNA polymerase for cDNA amplification

The whole RT reaction can be directly amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB #M0541) or Q5 Hot Start High-Fidelity 2X Master Mix (NEB #M0494). It can also be directly amplified with other commonly used polymerases or PCR master mixes described in publications [3-6].

References:

  1. Kapteyn, J. et al (2010) BMC Genomics, 11:413.
  2. Truchinovich, A. et al (2014) RNA Biol. 11, 817-828.
  3. Picelli, S. et al. (2013) Nat. Methods, 10, 1096-1098.
  4. Picelli, S. et al. (2014) Nat. Protoc. 9, 171-181.
  5. Soumilllon et al. (2014) bioRxive 10.1101/003236.
  6. Bagnoli, JW. et al (2018) Nat Commun. 9 (1):2937.