TITLE: Synthesis of in vitro transcribed RNA from whole bacterial transcriptome
AUTHORS: Bhargava   Reddy Morampalli, Olin  Silander, Bhargava   Reddy Morampalli, Olin  Silander

[DESCRIPTION]
Identifying RNA modifications across the transcriptome has been made possible by direct RNA sequencing using nanopore sequencing from Oxford Nanopore Technologies. This is due to voltage changes happening when each nucleotide passes through the pore. Hypothesis is that since each nucleotide passing through the pore has a distinct change in voltage, nucleotides with epigenetic modifications (e.g.methylation) also has distinct voltage changes when passing through. This is identified by errors in basecalling.

Several software have been developed to identify locations of modifications using machine learning, deep learning models. In order to make highly accurate predictions, there is the necessity of comparing the signals from native RNA sequencing with RNA without any modifications. A few recent research publications get RNA through in vitro transcription but only for ribosomal RNAs (rRNA). This is easier since there are only 7 different ribosomal RNAs for each type i.e., 16S, 23S and 5S. 

Getting IVT RNA for entire transcriptome is not trivial since bacterial transcriptome is complex and depends on the condition the bacteria has been grown in. We use the method of strand switching during cDNA synthesis to get cDNA for whole bacterial transcriptome which can then be used as a template for IVT.

[BEFORE_START]
Clean the bench surfaces and pipettes using RNase ZAP to inhibit all RNases.
Always use RNase free centrifuge tubes, PCR tubes and pipette tips for all steps wherever required.

[GUIDELINES]
Great precautions should be taken when handling RNA.

[STEPS]
SECTION: Addition of poly(A) tail to E. coli RNA
1. A reaction is setup in a PCR tube using the following components


 


SECTION: Addition of poly(A) tail to E. coli RNA
2. Incubate the reaction mixture for 15 min  at 37 °C in a thermocycler followed by 20 min  at 65 °C for inactivation.

SECTION: Addition of poly(A) tail to E. coli RNA
3. Transfer the reaction mixture into a RNase free tube for cleanup step. Add 45 µL  of RNAClean AMPure XP beads and 105 µL of 100 % ethanol to the reaction mixture

SECTION: Addition of poly(A) tail to E. coli RNA
4. Flick the tube for mixing and incubate at Room temperature for 5 min .

SECTION: Addition of poly(A) tail to E. coli RNA
5. Keep the tube on a magnetic rack and allow the beads to pellet towards the magnet
SECTION: Addition of poly(A) tail to E. coli RNA
6. Prepare 500 µL of 80% ethanol during this time. Pipette the solution out of the tube taking care to not disturb the pellet

SECTION: Addition of poly(A) tail to E. coli RNA
7. Wash the beads by pipetting 150 µL of 80% ethanol and repeat the step. Remove the ethanol, spin and put the tube back in the magnetic rack to remove any left over solution

SECTION: Addition of poly(A) tail to E. coli RNA
8. Allow the beads to air dry for 30 s making sure not to over dry. Add 20 µL of RNase free water to the beads and incubate at Room temperature for 5 min to elute RNA.

SECTION: Addition of poly(A) tail to E. coli RNA
9. Place the tube back on the magnetic rack allowing the beads to separate and pipette the poly(A) tailed RNA in water into a new RNase free tube and keep it on ice  

SECTION: cDNA first strand synthesis
10. Prepare a reaction with the following components in a PCR tube


 

SECTION: cDNA first strand synthesis
11. Incubate the reaction mixture at 65 °C for 5 min in a thermocycler and then snap cool on a pre-chilled freezer block

SECTION: cDNA first strand synthesis
12. In a separate tube, mix together the following:

 
Reagent Volume    5x RT Buffer 4 μl   RNaseOUT 1 μl   Nuclease-free water 1 μl   Strand-Switching Primer (SSP) 2 μl   Total 8 μl
 
SECTION: cDNA first strand synthesis
13. Mix gently by flicking the tube, and spin down.
SECTION: cDNA first strand synthesis
14. Add the strand-switching buffer to the snap-cooled, annealed mRNA, mix by flicking the tube and spin down
SECTION: cDNA first strand synthesis
15. Incubate at 42 °C  for 2 min 

SECTION: cDNA first strand synthesis
16. Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 µl.
SECTION: cDNA first strand synthesis
17. Mix gently by flicking the tube, and spin down.
SECTION: cDNA first strand synthesis
18. Incubate using the following protocol:

 
Cycle step Temperature Time No. of cycles    Reverse transcription and strand-switching 42° C 90 mins 1   Heat inactivation 85° C 5 mins 1   Hold 4° C ∞ 
 
SECTION: RNA strand degradation and second strand synthesis
19. Add 1 µL  of RNase Cocktail Enzyme Mix (ThermoFisher, AM2286) to the reverse transcription reaction.

SECTION: RNA strand degradation and second strand synthesis
20. Incubate the reaction for 10 min  at 37 °C . 

SECTION: RNA strand degradation and second strand synthesis
21. Resuspend the AMPure XP beads by vortexing.
SECTION: RNA strand degradation and second strand synthesis
22. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: RNA strand degradation and second strand synthesis
23. Add 17 µL  of resuspended AMPure XP beads to the reaction and mix by flicking the tube.

SECTION: RNA strand degradation and second strand synthesis
24. Incubate on a Hula mixer (rotator mixer) for 5 min  at Room temperature .

SECTION: RNA strand degradation and second strand synthesis
25. Prepare 500 µL  of fresh 70% ethanol in nuclease-free water.

SECTION: RNA strand degradation and second strand synthesis
26. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: RNA strand degradation and second strand synthesis
27. Keep the tube on the magnet and  wash the beads with 200 µL  of freshly prepared 70% ethanol without  disturbing the pellet. Remove the ethanol using a pipette and discard.


SECTION: RNA strand degradation and second strand synthesis
28. Repeat the previous step.
SECTION: RNA strand degradation and second strand synthesis
29. Spin down  and place the tube back on the magnet. Pipette off any residual  ethanol. Allow to dry for ~30 s, but do not dry the pellet to the point of cracking.
SECTION: RNA strand degradation and second strand synthesis
30. Remove the tube from the magnetic rack and resuspend pellet in 20 µL  nuclease-free water.

SECTION: RNA strand degradation and second strand synthesis
31. Incubate on a Hula mixer (rotator mixer) for 10 min  at Room temperature .

SECTION: RNA strand degradation and second strand synthesis
32. Pellet beads on magnet until the eluate is clear and colourless.
SECTION: RNA strand degradation and second strand synthesis
33. Remove  and retain 20 µL  of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: RNA strand degradation and second strand synthesis
34. Prepare the following reaction in a 0.2 ml thin-walled PCR tube:

 
Reagent Volume    2x LongAmp Taq Master Mix 25 μl   PR2 Primer (PR2) 2 μl   Reverse-transcribed sample from above 20 μl   Nuclease-free water 3 μl   Total 50 μl
 
SECTION: RNA strand degradation and second strand synthesis
35. Incubate using the following protocol:

 
  Temperature Time No. of cycles    94 °C 1 mins 1   50 °C 1 mins 1   65 °C 15 mins 1   4 °C ∞ 
 
SECTION: RNA strand degradation and second strand synthesis
36. Resuspend the AMPure XP beads by vortexing.
SECTION: RNA strand degradation and second strand synthesis
37. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: RNA strand degradation and second strand synthesis
38. Add 40 µL  of resuspended AMPure XP beads to the reaction and mix by flicking the tube.

SECTION: RNA strand degradation and second strand synthesis
39. Incubate on a Hula mixer (rotator mixer) for 5 min  at Room temperature .

SECTION: RNA strand degradation and second strand synthesis
40. Prepare 500 µL  of fresh 70% ethanol in nuclease-free water.
SECTION: RNA strand degradation and second strand synthesis
41. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: RNA strand degradation and second strand synthesis
42. Keep the tube on the magnet and  wash the beads with 200 µL of freshly prepared 70% ethanol without  disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: RNA strand degradation and second strand synthesis
43. Repeat the previous step.
SECTION: RNA strand degradation and second strand synthesis
44. Spin down  and place the tube back on the magnet. Pipette off any residual  ethanol. Allow to dry for ~30 s, but do not dry the pellet to the  point of cracking.

SECTION: RNA strand degradation and second strand synthesis
45. Remove the tube from the magnetic rack and resuspend pellet in 21 µl nuclease-free water.
SECTION: RNA strand degradation and second strand synthesis
46. Incubate on a Hula mixer (rotator mixer) for 10 min  at Room temperature .
SECTION: RNA strand degradation and second strand synthesis
47. Pellet beads on magnet until the eluate is clear and colourless.
SECTION: RNA strand degradation and second strand synthesis
48. Remove and retain 21 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: RNA strand degradation and second strand synthesis
49. Measure the concentration of the double stranded cDNA obtained after this step.
SECTION: in vitro transcription
50. 100 ng of double stranded cDNA synthesized is used for in vitro transcription.
SECTION: in vitro transcription
51. New England Biolab's HiScribe™ T7 Quick High Yield RNA Synthesis Kit (E2050s) is used for RNA synthesis using cDNA as a template.
SECTION: in vitro transcription
52. Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Keep on ice. 
SECTION: in vitro transcription
53. 
Assemble the reaction at room temperature in the following order: 

 

SECTION: in vitro transcription
54. Mix thoroughly by pipetting and pulse-spin in a microfuge.
SECTION: in vitro transcription
55. Incubate at  37 °C  for 120 min . Incubation over 2 hours will no negatively affect the RNA synthesis

SECTION: in vitro transcription
56. 30 µL  of Nuclease-free water to the reaction mixture and 2 µL of DNase I (included in the kit) is added after that.
SECTION: in vitro transcription
57. Mix the reaction mixture by pipetting  and incubate for 15 min at 37 °C 

SECTION: in vitro transcription
58. Following the incubation, RNA is purified using any RNA cleanup kit. I used the RNA Clean & Concentrator-5 kit (R1013) and finally eluted in 20 µL of RNase-free water.

SECTION: in vitro transcription
59. Concentration is checked using Qubit RNA kit.