TITLE: In vitro transcription, capping, and 2'-O methylation of long RNAs
AUTHORS: Stephen Floor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for in vitro transcription of long RNAs off plasmid or PCR product templates. It is assumed that the template has been gel purified, is the right size, has a T7 promoter and a 3' polyA tail (typically A60). Following this protocol the RNA will be ready for transfection into mammalian cells or in vitro translation. Significant amounts of template are necessary: at least 1ug of template per 100ul reaction; ideally closer to 5ug of template, especially for  very long templates. Use RNase sensitive protocols and reagents for all steps of this procedure.   </div><div class = "text-block">The basic protocol is: </div><div class = "text-block">- PCR amplification of the template</div><div class = "text-block">- in vitro transcription</div><div class = "text-block">- capping and 2'-O-Methylation </div><div class = "text-block">- quality control and optional purification </div><div class = "text-block">Based on protocols from Kaihong Zhou (Doudna lab) and RNA: A Laboratory Manual (Rio, Ares, Hannon, Nilsen).</div></div>

[STEPS]
?. [PCR amplification of template]
Primer design: pA60 txn rev TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTG CAGpA60 txn fwd CGG CCA GTG AAT TCG AGC TCT AAT ACG ACT CAC TAT AGGnote: if using a different template, substitute the reverse and forward primers for primers that will amplify the forward and reverse of your template. The forward primer should have the T7 promoter in it. If the template does not have a genetically encoded polyA tail, as this one does, then your reverse primer will not have all these T's and instead you will have to polyA tail the RNAs with this: https://www.neb.com/products/m0276-ecoli-poly-a-polymerase
?. [PCR amplification of template]
PCR setup (4x 100ul for each template, four replicate reactions1):  AB1ReagentAmount per 100 ul reaction2HF buffer10 ul3dNTP (10 mM)2 ul4forward primer (100 uM)0.5 ul5reverse primer (100 uM)2 ul6phusion polymerase1 ul7template (50 ng total)variable8waterto 100 ul
AB1ReagentAmount per 100 ul reaction2HF buffer10 ul3dNTP (10 mM)2 ul4forward primer (100 uM)0.5 ul5reverse primer (100 uM)2 ul6phusion polymerase1 ul7template (50 ng total)variable8waterto 100 ul
?. [PCR amplification of template]
Run PCR with a two step protocol:    72 degrees annealing/extension for 3 minutes     98 degrees melting for 30 seconds    repeat 30 cycles    10 minute final extension time at 72
?. [PCR amplification of template]
Load all of the PCRs into an agarose gel and verify then size of the template band and then gel purify it
?. [In vitro transcription of RNA]
Warm all reagents except T7 polymerase and Superase:IN to room temperature and assemble the reaction at RT. warning: Adding cold spermidine to template can cause precipitation of the template.
?. [In vitro transcription of RNA]
Mix the following together in a 1.5ml tube  AB1Template DNA1 to 5 ug2ACGU rNTP mix (25 mM each, pH 8)30 ul310X transcription buffer (RT)10 ul41M DTT2 ul51M MgCl21 ul6T7 polymerase10 ul7Superase:IN1 ul8DEPC H2Oto 100 ul
AB1Template DNA1 to 5 ug2ACGU rNTP mix (25 mM each, pH 8)30 ul310X transcription buffer (RT)10 ul41M DTT2 ul51M MgCl21 ul6T7 polymerase10 ul7Superase:IN1 ul8DEPC H2Oto 100 ul
note: we typically use T7 polymerase we have purified but the Megascript kit in the materials or similar should also work. If using a kit, perform the in vitro transcription according to the kit instructions.
?. [In vitro transcription of RNA]
Incubate at 37 degrees C in a stable incubator - a water bath or stable dry incubator like a thermocycler. Warm rooms and heat blocks can have higher variability in temperature and T7 is a sensitive enzyme.
?. [In vitro transcription of RNA]
Wait 1 hour and check the reactions - they should be somewhat cloudy. If they are, add 1 ul of 1 mg/ml pyrophosphatase to each (Roche 10108987001; optional)
?. [In vitro transcription of RNA]
Allow the reactions to proceed for 3-4 hours.
?. [In vitro transcription of RNA]
Add 1 ul of RQ1 RNase-free DNase to each reaction and incubate at 37 for 30 minutes.
?. [In vitro transcription of RNA]
Precipitate by adding 200 ul 0.3M NaOAc pH 5.2, 1 ul GlycoBlue and 750 ul 100% EtOH.
?. [In vitro transcription of RNA]
[PAUSE] Incubate at -80 for >1 hour to overnight.
?. [Resuspending EtOH precipitated RNA]
Pellet precipitations by centrifugation at 16,000 g at 4 degrees for 25 minutes.
?. [Resuspending EtOH precipitated RNA]
Aspirate supernatant and discard
?. [Resuspending EtOH precipitated RNA]
Wash with 500 ul -20 degree 70% EtOH
?. [Resuspending EtOH precipitated RNA]
Centrifuge at 16,000 g and 4 degrees for 5 minutes
?. [Resuspending EtOH precipitated RNA]
Aspirate supernatant and discard
?. [Resuspending EtOH precipitated RNA]
Quick-spin and remove residual ethanol with a P20 pipet
?. [Resuspending EtOH precipitated RNA]
Allow pellet to air dry for 5 minutes; do not overdry
?. [Resuspending EtOH precipitated RNA]
Resuspend pellet in 100 ul 50 mM EDTA
?. [Resuspending EtOH precipitated RNA]
Purify RNA over Zymo RNA Clean & Concentrator 25 column following small RNA removal protocol and elute in 30 ul DEPC H2O
?. [Resuspending EtOH precipitated RNA]
Recommended but optional: assess RNA integrity by running a Glyoxal gel before proceeding
?. [Resuspending EtOH precipitated RNA]
Measure concentration of RNA on nanodrop
?. [Capping and 2'-O-Methylation]
Dilute 20 ug of RNA in 26 uL DEPC H2O
?. [Capping and 2'-O-Methylation]
Prepare a 4mM stock of SAM from the supplied 32 mM stock (4 uL 32mM and 28 uL DEPC H2O for up to 15 reactions)
note: SAM is unstable and should not be freeze-thawed. If the SAM is degraded, there will not be mRNA capping or methylation.
?. [Capping and 2'-O-Methylation]
Either add these directly to the tube above, or assemble a master mix according to the following recipe if performing many reactions (note all these reagents are supplied by the corresponding kits):  AB1ReagentVolume per 40 ul reaction210X capping buffer4 ul310 mM GTP2 ul44 mM SAM2 ul5Vaccinia capping enzyme2 ul62'-O-Me transferase2 ul7RNasin plus (Promega)2 ul
AB1ReagentVolume per 40 ul reaction210X capping buffer4 ul310 mM GTP2 ul44 mM SAM2 ul5Vaccinia capping enzyme2 ul62'-O-Me transferase2 ul7RNasin plus (Promega)2 ul
?. [Capping and 2'-O-Methylation]
Add 14 uL of the master mix to each tube with RNA
?. [Capping and 2'-O-Methylation]
Incubate at 37 deg for 60 minutes
?. [Capping and 2'-O-Methylation]
Purify over zymo Clean and Concentrator 25 column following small RNA removal protocol and elute in 30 ul DEPC H2O
if your RNA is not A-tailed from the PCR template and you want it to have a polyA tail, you should A-tail it with E. coli poly-A polymerase at this stage. Follow the kit instructions in materials
?. [Quality control: glyoxal gel]
Run a glyoxal gel of all RNAs and verify their size.
?. [Quality control: glyoxal gel]
Also glyoxylate the ladder and include (typically I use 5ul of either of the ladders listed above)
?. [Quality control: glyoxal gel]
Use Image Lab to quantify the full-length band and use this to determine concentration in combination with nanodrop or qubit readings.
?. [Quality control: glyoxal gel]
If significant truncated products are present I recommend agarose gel purification of full-length RNA using the Zymo Agarose Gel Zymoclean kit (R1011)