This method can help answer key questions in the field of transcriptional mutagenesis such as which RNA polymerase subunits or biological processes control the fidelity of transcription. The main advantage of this technique is that it allows for the measurement of endogenous transcription errors in the transcriptomes of eukaryotic organisms. Generally, individuals new to this protocol will struggle because the length and complexity of the assay and a need for advanced bioinformatics in order to interpret the data.
To begin the protocol, circularize the RNA fragments by heat denaturing 20 microliters of the previously prepared sample at 65 degrees Celsius for one minute. After one minute, immediately place the sample on ice for two minutes. Next, add four microliters of 10x T4 RNA ligase buffer, four microliters of 10 milliMolar ATP, one microliter of RNase inhibitor, one microliter of nuclease free water, and eight microliters of 50%polyethylene glycol, or PEG.
Then mix the sample thoroughly by vortexing. Then add two microliters of 10 units per microliter of T4 RNA ligase one. Mix the sample again by pipetting and incubate it at 25 degrees Celsius for two hours in a thermocycler with the lid temperature set at 30 degrees Celsius.
After the two hour incubation, add 10 microliters of nuclease free water to the sample and clean the sample with an oligo cleanup and concentration kit. Elute the sample in 20 microliters of nuclease free water. To reverse transcribe the circular RNA molecules, add four microliters of 10 milliMolar dNTPs, four microliters of 50 nanograms per microliter random hexamers, and nine microliters of nuclease free water to nine microliters of RNA.
Mix thoroughly by pipetting and denature the sample at 65 degrees Celsius for one minute. After denaturing the sample, place it on ice for two minutes. Add eight microliters of 5x first strand synthesis buffer, two microliters of 0.1 milliMolar DTT, and four microliters of 200 units per microliter reverse transcriptase to the sample and mix by pipetting.
Incubate the sample at 25 degrees Celsius for 10 minutes with the lid set five degrees Celsius higher than the incubation temperature. Then incubate the sample for 20 minutes at 42 degrees Celsius with the lid set at 47 degrees Celsius. Elute the sample in 42 microliters of elution solution after cleaning up with the clean up and concentrator kit.
Use the second strand synthesis kit to generate a double-stranded cDNA library. Place the samples on ice and add 30 microliters of NF water to 38 microliters of your sample. Next, add eight microliters of 10x second strand buffer and four microliters of second strand enzyme and mix the sample by gentle pipetting before incubating at 16 degrees Celsius for two and a half hours.
Clean up the samples with the oligo clean up and concentration kit for 100 microliter reactions and elute the samples in 38 microliters of nuclease free water. Mix the end repair reaction by pipetting and incubate it at 20 degrees Celsius for 30 minutes. Follow the 20 degree Celsius incubation with a 30 minute incubation at 65 degrees Celsius.
First, dilute the adapters for next generation sequencing ten-fold in 10 milliMolar tris HCl to a final concentration of 1.5 microMolar. Then add 2.5 microliters of diluted adapter and one microliter of ligation enhancer to each sample and mix them by vortexing. Next, add 15 microliters of blunt TA ligase master mix.
Pipette the sample up and down to mix and incubate it at 20 degrees Celsius for 15 minutes. Then, add three microliters of uracil specific excision reagent enzyme and incubate the sample at 37 degrees Celsius for 15 minutes. After the ligation is complete, add 13.5 microliters of nuclease free water to the sample for a total volume of 100 microliters and proceed to size selection.
Acclimate magnetic beads to room temperature. Add 30 microliters of acclimated, resuspended magnetic beads to each sample and mix by pipetting. Transfer the sample to a 1.5 milliliter tube and incubate it at room temperature for five minutes.
Place the sample on a magnetic stand for five minutes to separate the beads from the supernatant. Transfer the supernatant to a new 1.5 milliliter tube and dispose of the magnetic beads. Add a fresh aliquot of 15 microliters of magnetic beads to each sample.
Mix thoroughly by pipetting and incubate for five minutes at room temperature. Place the samples on a magnetic rack and incubate them for five minutes at room temperature. Then carefully remove and dispose of the supernatants, making sure not to disturb the beads.
Add 200 microliters of 80%freshly prepared ethanol to each of the samples without disturbing the pelleted magnetic beads and incubate for 30 seconds. Then, fully remove any trace of ethanol from the samples and air dry the beads for five minutes, but be careful not to over-dry the beads. To elute the samples from the beads, remove the tubes from the magnetic stand.
Add 19 microliters of 10 milliMolar tris HCl. Pipette the mix up and down multiple times to resuspend the beads and incubate the samples for five minutes at room temperature. Place the samples back on the magnetic stand and incubate them for five minutes to separate the beads from the supernatant.
Carefully remove 15 microliters of the supernatant containing the purified, size-selected cDNA libraries from the tubes and transfer it to a fresh 1.5 milliliter tube. Add five microliters of universal primer and five microliters of a unique index primer to each purified cDNA library and mix them thoroughly by pipetting. Carefully check the polymerase master mix for crystals and other precipitates and warm the master mix by hand until the precipitates dissolve.
Add 25 microliters of the polymerase master mix to the sample. Mix the sample by pipetting and follow the cycling conditions listed in the accompanying text protocol for the PCR amplification. Clean up the final libraries by adding 45 microliters of resuspended magnetic beads directly to the PCR reaction.
Mix them by pipetting and incubate them at room temperature for five minutes. Transfer the sample to a 1.5 milliliter tube and place it in a magnetic stand for five minutes. Following incubation, discard the supernatant and wash it twice with freshly prepared 80%ethanol for 30 seconds.
After the second wash, fully remove the ethanol from the sample and air dry the bead pellet for five minutes. Then elute it in 35 microliters of 0.1x TE.Resuspend the beads by pipetting them and incubate them at room temperature for five minutes. After the tube has been on the magnetic stand for five minutes, transfer 30 microliters of the supernatant to a fresh 1.5 milliliter tube.
Store the libraries at minus 80 degrees Celsius. After isolation of the RNA, two distinct rRNA peaks are visible on the electrophoretogram. Fragmentation of the RNA will result in 50 to 70 base pair RNA fragments.
Rolling circle reverse transcription was used to generate cDNA molecules that are comprised of at least three tandem repeats of the circular RNA templates. Size-selection with magnetic beads allows libraries of the appropriate size to be purified. Sequencing information of a single C-seq library shows that most repeats tend to be 45 to 80 bases in length.
Approximately 50%of the bases that were sequenced are part of these repeats. Since most of these bases are present in rates that contain three repeats or more, the number of unique bases that are sequenced is about one third of the total number of bases sequenced. Once it has been mastered, this technique can be done in two to three days if it is performed properly.
While attempting this procedure, it is important to always maintain a sterile working environment, free of RNAses that could interfere with your work. It is also important to carefully follow each step to ensure that no mistakes are made during the procedure. After watching this video, you should have a good understanding of how to measure transcription errors in eukaryotes using the C-seq assay.
