The overall goal of this method is to capture and sequence mRNA three prime ends, thereby, enabling studies of mRNA processing, particularly three prime end cleavage and polyadenylation, as well as the quantification of gene expression. The A-seq2 protocol and the data analysis package presented here, can help to answer key questions about the generation and function of transcript isoforms in different cell types and tissues. The main advantages of the A-seq2 method are that it avoids sequencing through long poly(A)stretches and it does not generate adapter dimers.

Cells that are grown to 80%confluency are used for this procedure. Remove the growth medium and wash the cells once with PBS. Directly lyse the cells on the plate by adding one milliliter of lysis buffer per well and using a rubber spatula to completely detach the cell material from the plate surface.

Use a one milliliter pipette tip to transfer the viscous lysate from each well into a 15 milliliter plastic tube. Share the DNA with a one milliliter syringe attached to a 23 gauge hypodermic needle. Perform several vigorous up and down movements of the plunger, until the lysate is no longer viscous.

Transfer the lysate into a 1.5 milliliter tube. Spin at 20, 000 times g and four degrees Celsius for five minutes, to remove the debris. Collect the clear supernatant from the lysate.

Add to oligo d(T)25 magnetic beads and resuspend the mixture. Place the tubes on a rotating wheel for 10 minutes. Next, place the tubes on a magnetic rack for two minutes.

Remove the clear liquid. Add 0.8 milliliters of buffer A to each tube and turn the tube by 180 degrees two to three times. Remove buffer A from the second wash.

Add 0.8 milliliters of buffer B to each tube and leave on the rack for two minutes. To elute the bound mRNA from the beads, remove buffer B, add 33 microliters of water to each tube, and resuspend the beads. Heat to 75 degrees Celsius for five minutes on a hearted block.

Immediately, spin the tubes for one second and place them on the magnetic rack. Transfer the supernatant to a new tube. Add 66 microliters of alkaline hydrolysis buffer to each tube, mix and heat at 95 degrees Celsius for exactly five minutes on a heating block.

Immediately, chill the tubes on ice. Subsequently, perform RNA isolation using an RNA cleanup kit, as described in the text protocol. To begin this procedure, add to each RNA sample 14 microliters of a polynucleotide kinase Master Mix.

Incubate at 37 degrees Celsius, for 30 minutes. After 30 minutes, load the kinase reactions onto prepared spin columns. Spin the columns at 735 times g for two minutes.

Discard the columns and place the tubes with collected reactions on ice. To block the three prime ends of RNA fragments to avoid their concatemerization in subsequent ligation reactions, add to each sample 17.5 microliters of a poly(A)polymerase Master Mix. Mix and spin for one second.

Incubate at 37 degrees Celsius for 30 minutes. After 30 minutes, add 32.5 microliters of water to each reaction and purify the RNA, as described in the text protocol. Start this procedure by placing the reactions in a vacuum concentrator for 10 minutes, to reduce the volume to six microliters.

Then add to each reaction 24 microliters of an RNA ligase Master Mix. Incubate the reactions on a heated mixer at 24 degrees Celsius for 16 hours, with intermittent mixing at one thousand RPM. On the following day, add 17 microliters of water to each reaction and mix.

Purify the RNA, as described in the text protocol. Place the eluites in a vacuum concentrator for three minutes, to reduce the volume to 11 microliters. Transfer the reactions to 200 microliter PCR tubes for the reverse transcription reaction.

Add one microliter of primer. Heat at 70 degrees Celsius in a PCR cycler for five minutes and then leave at room temperature for five minutes. Add eight microliters of a reverse transcription Master Mix to each tube and mix.

Put the tubes in a PCR cycler. Heat to 55 degrees Celsius for 10 minutes and to 80 degrees Celsius for another 10 minutes. Keep on ice.

Prepare streptavidin beads, as described in the text protocol. Add the reverse transcription reaction to the bead solution and incubate at four degrees Celsius on a rotating wheel for 20 minutes. Using a magnetic rack, wash the beads twice with biotin binding buffer and twice with TEN buffer.

Resuspend the beads in 50 microliters of TEN buffer. Add two microliters of uracil DNA glycosylase enzyme mix and incubate at 37 degrees Celsius for one hour in a mixer, with intermittent mixing. Add 50 microliters of water, 11 microliters of RNase H buffer and one microliter of Rnase H, to each reaction.

Incubate at 37 degrees Celsius for 20 minutes. Place the tubes on the magnetic rack and transfer the liquid containing the cleaved cDNA to a new tube. Purify the cleaved cDNA using a PCR purification kit, as described in the text protocol.

Place the purified cDNA in a vacuum concentrator for eight minutes to concentrate to a volume of seven microliters. To ligate five prime adapters to the five prime ends of the isolated cDNA, add to each sample 23 microliters of an RNA ligase Master Mix and incubate at 24 degrees Celsius for 20 hours. Lastly, add 70 microliters of water to each reaction.

A pilot PCR is performed to determine the optimal number of PCR cycles to reach library amplification, within the exponential phase. Pipette the following into a 200 microliter PCR tube:25 microliters of DNA polymerase mix, 20 microliters of ligation reaction, two microliters of water, 1.5 microliter of forward PCR primer and 1.5 microliter of reverse PCR index primer. Run the cycler with the following program:three minutes at 95 degrees Celsius, followed by 20 cycles of 20 seconds at 98 degrees Celsius, 20 seconds at 67 degrees Celsius and 30 seconds at 72 degrees Celsius.

Collect seven microliter aliquots directly from the cycler, after the indicated cycles. Separate the products on a 2%agarose gel and visualize migration of PCR products. Determine the number of cycles at the beginning of exponential amplification, 12 cycles in this example, and use this number of cycles for a large scale PCR reaction.

Separate the products from the large scale PCR reaction on a 2%agarose gel and cut out the gel slices containing 200 to 350 nucleotide DNA products. Subsequently, extract the DNA from the gel slices with the gel extraction kit, as described in the text protocol. Only the most important computational steps will be shown in this video.

More details are available in the text protocol. Start by cloning the Git repository and changing to the newly created directory. Create an environment that contains the software and activate this environment.

Download the genome sequence of the organism, from which A-seq2 data has been obtained. Open the configuration file and set the input parameters, as indicated in the text protocol. Start the analysis.

The response of a specific gene, NUP214 to the knockdown of the HNRNPC protein, was examined by analyzing a-seq2 reads from two samples of HEK-293 cells, treated either with a control small interfering RNA or with a HNRNPC small interfering RNA. The reads that documented poly(A)sites annotated by the analysis pipeline were saved in BAM format, that was used as input to the IGD genome browser. The three prime ends of the read peaks, mapped at mMRNA three prime ends that are annotated in ensemble.

The profiles indicate an increased use of the long three prime UTR isoform upon HNRNCR knockdown. Once mastered, the A-seq protocol takes eight hours of hands-on time or about two to three days, not counting the time for the cell culture and the overnight ligations. When attempting the protocol, it's important to first design a primal set that conforms to sequencing platform used at your location.

Once you obtained mMRA three prime ends, other methods, like cross linking and immunoprecipitation can be preformed, to identify regulators of pre-mRNA three prime end processing. A-seq2 paved the way for researchers in the field of RNA processing, to explore the regulation and consequences of alternative polyadenlylation in individual cell types. After watching this video, you should have a good understanding on how to generate libraries of mRNA three prime ends, analyze your sequencing data, identify new poly(A)sites and quantify your usage.

We hope A-seq2 will ease your work in starting the regulation of mRNA processing and lead to many new insights.