Oct 15, 2021
Synthesis of in vitro transcribed RNA from whole bacterial transcriptome
- 1Massey University
Protocol Citation: Bhargava Reddy Morampalli, Olin Silander, Bhargava Reddy Morampalli, Olin Silander 2021. Synthesis of in vitro transcribed RNA from whole bacterial transcriptome. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb7r7yvpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 10, 2021
Last Modified: October 15, 2021
Protocol Integer ID: 46157
Keywords: in vitro transcription, direct RNA sequencing, RNA modifications
Abstract
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.
Guidelines
Great precautions should be taken when handling RNA.
Materials
HiScribe™ T7 Quick High Yield RNA Synthesis Kit - E2050S
RNA Clean & Concentrator-5 - R1013
RNaseOUT
Primers: oligo-d(T)-22 primer, T7-Strand Switch Primer, PR2/T7 promoter primer (primer sequences commented in the protocol)
10 mM dNTP solution
Nuclease-free water (Thermofisher - #AM9937)
0.2 mL thin-walled PCR tubes
1.5 mL Eppendorf LoBind tubes
Maxima H Minus Reverse Transcriptase (200 U/uL) with 5x RT buffer (Thermofisher - #EP0751)
Pre-chilled freezer block at -20 C
LongAmp Taq 2X MasterMix (NEB - #M0287)
RNase Cocktail Enzyme Mix (Thermofisher - #AM2286)
RNAclean Agencourt XP beads
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.
Addition of poly(A) tail to E. coli RNA
Addition of poly(A) tail to E. coli RNA
A reaction is setup in a PCR tube using the following components
| A | B | C | D | E | F | G | H | |
| Components | Volume (µL) | |||||||
| RNA | 1 - 10 µg | |||||||
| 10x E. coli poly(A) polymerase reaction buffer | 2 | |||||||
| ATP (10 mM) | 2 | |||||||
| RNase inhibitor | 1 | |||||||
| E. coli poly(A) polymerase | 1 | |||||||
| Water | upto 25 µL | |||||||
| Total | 25 |
Incubate the reaction mixture for 00:15:00 at 37 °C in a thermocycler followed by 00:20:00 at 65 °C for inactivation.
35m
Transfer the reaction mixture into a RNase free tube for cleanup step. Add 45 µL (1.8 volumes of reaction) of RNAClean AMPure XP beads and 105 µL (4.2 volumes) of 100 % ethanol to the reaction mixture
Flick the tube for mixing and incubate at Room temperature for 00:05:00 .
5m
Keep the tube on a magnetic rack and allow the beads to pellet towards the magnet
Prepare 500 µL of 80% ethanol during this time. Pipette the solution out of the tube taking care to not disturb the pellet
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
Allow the beads to air dry for 00:00:30 making sure not to over dry. Add 20 µL of RNase free water to the beads and incubate at Room temperature for 00:05:00 to elute RNA.
5m 30s
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
cDNA first strand synthesis
cDNA first strand synthesis
1h 45m
1h 45m
Prepare a reaction with the following components in a PCR tube
| A | B | |
| Reagent | Volume | |
| poly A+ RNA | 1 µg (x µL) | |
| anchored oligo d(T)22 | 1 µL | |
| 10 mM dNTPs | 1 µL | |
| RNase free water | 9 - x µL | |
| Total | 11 µL |
Incubate the reaction mixture at 65 °C for 00:05:00 in a thermocycler and then snap cool on a pre-chilled freezer block
5m
In a separate tube, mix together the following:
| A | B | |
| Reagent | Volume | |
| 5x RT Buffer | 4 μl | |
| RNaseOUT | 1 μl | |
| Nuclease-free water | 2.5 μl | |
| T7 - Strand-Switching Primer (SSP) | 0.5 µL | |
| Total | 8 μl |
Mix gently by flicking the tube, and spin down.
Add the strand-switching buffer to the snap-cooled, annealed mRNA, mix by flicking the tube and spin down
Incubate at 42 °C for 00:02:00
2m
Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 µl.
Mix gently by flicking the tube, and spin down.
Incubate using the following protocol:
| A | B | C | D | |
| 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 | ∞ |
1h 35m
RNA strand degradation and second strand synthesis
RNA strand degradation and second strand synthesis
58m
58m
Add 1 µL of RNase Cocktail Enzyme Mix (ThermoFisher, AM2286) to the reverse transcription reaction.
Incubate the reaction for 00:10:00 at 37 °C .
10m
Resuspend the AMPure XP beads by vortexing.
Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 17 µL of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at Room temperature .
5m
Prepare 500 µL of fresh 70% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
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.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend pellet in 20 µL nuclease-free water.
Incubate on a Hula mixer (rotator mixer) for 00:10:00 at Room temperature .
10m
Pellet beads on magnet until the eluate is clear and colourless.
Remove and retain 20 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Prepare the following reaction in a 0.2 ml thin-walled PCR tube:
| A | B | |
| Reagent | Volume | |
| 2x LongAmp Taq Master Mix | 25 μl | |
| PR2 Primer (PR2) - T7 promoter primer (IDT) | 3.75 μl | |
| Reverse-transcribed sample from above | 20 μl | |
| Nuclease-free water | 1.25 μl | |
| Total | 50 μl |
Incubate using the following protocol:
| A | B | C | |
| Temperature | Time | Cycle | |
| 94 °C | 1 mins | 1 | |
| 50 °C | 1 mins | 1 | |
| 65 °C | 15 mins | 1 | |
| 4 °C | ∞ |
17m
Resuspend the AMPure XP beads by vortexing.
Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 40 µL of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 00:05:00 at Room temperature .
5m
Prepare 500 µL of fresh 70% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
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.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend pellet in 21 µl nuclease-free water.
Incubate on a Hula mixer (rotator mixer) for 00:10:00 at Room temperature .
10m
Pellet beads on magnet until the eluate is clear and colourless.
Remove and retain 21 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Measure the concentration of the double stranded cDNA obtained after this step.
in vitro transcription
in vitro transcription
2h 15m
2h 15m
100 ng of double stranded cDNA synthesized is used for in vitro transcription.
New England Biolab's HiScribe™ T7 Quick High Yield RNA Synthesis Kit (E2050s) is used for RNA synthesis using cDNA as a template.
Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Keep on ice.
Assemble the reaction at room temperature in the following order:
| A | B | |
| Components | Volume | |
| Nuclease-free water | used to make up the volume to 20 μL | |
| NTP Buffer Mix | 10 μL (10 mM each NTP final) | |
| Template DNA (cDNA) | X μL (100 ng - 1 μg) | |
| T7 RNA Polymerase Mix | 2 μL | |
| Total Reaction Volume | 20 μL |
Mix thoroughly by pipetting and pulse-spin in a microfuge.
Incubate at 37 °C for 02:00:00 . Incubation over 2 hours will no negatively affect the RNA synthesis
2h
30 µL of Nuclease-free water to the reaction mixture and 2 µL of DNase I (included in the kit) is added after that.
Mix the reaction mixture by pipetting and incubate for 00:15:00 at 37 °C
15m
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.
Concentration is checked using Qubit RNA kit.