Protocol for in vitro transcribing mRNAs with defined poly(A)-tail lengths and visualizing sequential PABP binding

Summary Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails. We detail quality control steps throughout the procedure. For complete details on the use and execution of this protocol, please refer to Grandi et al.1


SUMMARY
Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation.Biochemical assays offer significant advantages for this purpose.Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach.We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails.We detail quality control steps throughout the procedure.For complete details on the use and execution of this protocol, please refer to Grandi et al. 1

BEFORE YOU BEGIN
3][4][5] In differentiated cells, PABPs are typically present in excess compared to mRNAs. 3,6,7Under such non-limiting conditions, it is believed that the poly(A)tail length and the number of PABP units bound to it correlate. 7,8re we describe how to in vitro synthesize mRNAs with different and precise poly(A)-tail lengths, followed by the visualization of the sequential binding of PABPs using capillary electrophoresis.As purified proteins are notably expensive, our protocol has the advantage of minimizing the volumes used for the binding and the mobility shift assay.
Note: While this protocol describes the synthesis of GFP-coding mRNAs, the reporter gene sequence can be customized depending on the application.The synthesized mRNAs can be further processed (i.e. 5 0 capped) and can be transfected into cells for follow-up analysis.While we have only focused on the in vitro binding of the mRNAs to PABPC1, the protocol should nevertheless work for other RNA Binding Proteins (RBPs) of interest, though this might require further optimization.
CRITICAL: All steps of the protocol should be performed with RNase-free reagents to prevent RNase contamination.
1. Choose the DNA template that includes the sequence of the reporter gene of your interest.
Note: This can be a plasmid, PCR product, cDNA, genomic DNA, or synthetic DNA.In most cases, the templates do not contain the T7 promoter sequence required by the T7 polymerase to start transcription.This sequence can be added to the DNA template through a PCR reaction using specific primers, as described below.Here, we use the pEF-GFP 9 plasmid as a starting point.

Design of primers for the DNA template
Timing: 1 h 2. Design a forward primer considering the following: a.If your template does not contain the T7 promoter, add it by designing a forward primer containing its sequence.
Note: Table 1 reports an example of a basic forward primer sequence containing an upstream spacer, T7 promoter, downstream spacer, Kozak sequence, start codon, and a gene specific sequence of approximately 17-22 nt.

CRITICAL:
The inclusion of all these sequences is essential for enhanced ribosome binding, transcription initiation and translation initiation, in case the mRNA has to be expressed in cells.
b.If your template already contains a T7 promoter and does not need the addition of any other regulatory sequence, design a 17-22 nt long forward primer specific for the beginning of your reporter gene.3. The reverse primers should anneal to the terminal region of the 3 0 UTR of your reporter gene and should contain a gene specific sequence of 17-22 nt, preceded by an oligo(dT) sequence of specified length (here we use 0, 5, 30, 50, 60, 100, 125 or 150 nt) at the 5 0 end.
See Table 2 for the primer sequences used by us.

Note:
We found that for poly(A)-tails longer than 150 nt, the PCR and IVT products show too many bands, suggesting that unspecific products are being synthesized.Furthermore, increasing the sequence length before the T7 promoter could increase transcription efficiency.We therefore suggest not to make tails longer than 150 nt, or to implement additional optimization strategies, and to test different 5 0 UTR sequences to determine the optimal yield.

Primer Sequence
Forward primer for the T7 promoter introduction 5 0 -GCTAATACGACTCACTATAGGGACAGGCCACCATGGTGAGCAAGGGCGAGGAGCTGT-3 0 Reverse primer for the introduction of defined poly(A)-tail lengths 5 0 -T(5/30/50/60/100/125/150)CCCATATGTCCTTCCGAGTG-3 0 Design of primers and linkers for the DNA and mRNA quality control In the section ''Quality control of the PCR products'' we describe how to check the purity of the poly(A)-tailed PCR products by sequencing.For this, a gene specific forward primer of 17-22 nt is needed.

Note:
The primer should bind 200-300 nt upstream to the poly(A)-tail, to ensure proper sequencing of the latter.See Table 3 for the primer sequence used by us.

5.
To verify the purity of the poly(A)-tails on the mRNA products: a. Design a DNA linker that contains a PO 4 modification at the 5 0 end and a NH 2 modification at the 3 0 end and ligate it to the mRNA.

Note:
The 5 0 phosphate is needed to facilitate the ligation to the RNA, while the 3' NH 2 group prevents ligation of the linker to itself, and protects the linker from exonuclease activity, increasing the stability of the constructed molecule.
b.The ligated mRNA product is reverse transcribed with a reverse primer containing a complementary sequence to the linker.c.The obtained cDNA is then amplified using a forward primer of 17-22 nt binding approximately 300 nt upstream of the poly(A)-tail, along with the identical reverse primer employed during the reverse transcription process.d.Finally, the amplified cDNA can be sequenced using the same forward primer utilized in the PCR step.See Table 3 for the sequences used by us.

Preparation of buffers
Timing: 2 h 6.Before starting the protocol, make sure to have the buffers and solutions listed in the ''materials and equipment'' section ready.Mix the reagents and add bromophenol blue powder (a spatula tip is sufficient).Aliquot the solution and store at À20 C for a maximum of 1 year.Try to minimize freeze/thaw cycles.

KEY RESOURCES TABLE
CRITICAL: Formaldehyde and formamide are toxic when inhaled, ingested, or absorbed through the skin.Work in a fume hood and wear protective goggles.Bromophenol blue is recognized as environmentally hazardous.Handle it with caution, ensuring proper storage, ventilation, and personal protective equipment to minimize environmental impact and prevent harm.

Gel electrophoresis
CRITICAL: EtBr is a potent mutagen and therefore needs to be handled with care, wearing protective clothing and gloves.
Alternatives: Safer and non-mutagenic alternatives to Ethidium Bromide are SYBR Green, GelRed, GelGreen and EvaGreen.It is also possible to analyze the PCR products by capillary electrophoresis using Agilent DNA Kits (Bioanalyzer).

STEP-BY-STEP METHOD DETAILS
Addition of T7 promoter and poly(A)-tail sequence to the DNA template The pEF-GFP plasmid is amplified with a forward primer that allows the incorporation of the T7 promoter sequence and a reverse primer with variable poly(dT) sequences (see Table 2 for the sequences used by us).This allows the incorporation of poly(A)-tails of defined lengths to the DNA template.
1. Thaw all reagents at RT and keep on ice.
2. Add to a 0.2 mL tube the PCR components, as specified in Table 4. Set up the reaction on ice.CRITICAL: For in-house purified DNA polymerases it is important to determine the enzyme activity prior use (defined as the amount of enzyme required to incorporate 1 nmol of nucleotides per minute).We have determined the activity of our enzyme by measuring the incorporation of fluorescently labelled nucleotides, but this can also be calculated through spectrophotometric assays, by measuring changes in absorbance due to the incorporation of nucleotides or the production of pyrophosphate.
3. Incubate the reaction in a thermocycler with pre-heated lid (95 C) following the conditions reported in Table 5.
Note: 30 s of annealing time is a consensus time.Longer or shorter times can be applied depending on the behavior of the primers or PCR yield.
Note: Cycle elongation time can vary depending on the length of the DNA that is amplified.Generally, use 1 min per 1000 bp for the Pfu DNA polymerase.
Note: We used 5 C per second as ramp time between the steps, but also lower speeds can be used.
Note: Over-cycling may result in undesired side reactions, especially impacting the accuracy of the poly(A)-tail length.In fact, the misalignment of the tailed reverse primer to the newly synthesized tailed products can lead to the synthesis of PCR products with unspecific poly(A)-tail length, which will get further amplified in the following cycles.

Note:
The amount of template reported in the table is optimized for the pEF-GFP plasmid.This can vary depending on the DNA template used:  Note: Any alternative purification method is good as long as the final PCR products are free from contaminants that could potentially disrupt following reactions.
a. Perform all centrifugation steps at 17900 g using a microcentrifuge at 20 C-22 C. 5. Quantify 1 mL of purified PCR product with a NanoDrop spectrophotometer: a. Select the protocol for dsDNA.b.Blank using the elution buffer used in step 4h.c.Add 1 mL of purified PCR product and measure the concentration.
CRITICAL: The A260/A280 and A260/A230 ratios are important to assess the purity of the samples.An A260/A280 ratio of approximately 1.8 indicates pure DNA.Lower values may imply contamination with proteins, phenol or other substances absorbing around 280 nm.The expected A260/230 ratio falls within the range 2.0-2.2.If ratios are below the expected ones, DNA samples should be re-purified by phenol/chloroform extraction in case of protein contaminants, or ethanol precipitation in case of the presence of small organic molecules.In case of RNA contamination, which contributes to the absorbance at 260 nm, samples should be treated with RNase.
Pause point: The resulting PCR products can be stored for multiple months at À20 C.

Quality control of the tailed PCR products
Timing: 2 h Before proceeding with IVT, it is recommended to check the quality of the tailed PCR products utilizing gel electrophoresis and DNA sequencing.Gel electrophoresis can highlight the presence of unspecific products and is crucial for verifying the expected size of the inserted poly(A)-tails.On the other hand, DNA sequencing ensures that the poly(A)-tails do not contain non-A nucleotides, which might be introduced by the DNA polymerase with different efficiencies depending on the poly(A)-tail length.
6. Make a 1.2% non-denaturing agarose gel: Note: The percentage of the agarose gel depends on the size of the expected PCR products.
a. Mix 250 ng of PCR products with 1 mL of TriTrack DNA Loading Dye (63) and add water up to 6 mL.b.Mix 3 mL of GeneRuler DNA Ladder Mix with 3 mL of water in a clean tube.c.Pipette the Ladder and the PCR products into the wells of the solidified gel.d.Run the gel in TBE 13 buffer using a voltage between 90-110 V until the DNA bands have migrated to the desired position.e. Remove the gel form the tray and inspect it on a UV transilluminator.f.The bands should be of the expected size (in this case 976 nt + the length of the added poly(A)tail) and the size should increase with the poly(A)-tail length (see Figure 1A in expected outcomes).7. Sequence the PCR products using a forward primer that binds at the 3 0 UTR and double-check that the poly(A)-tail does not contain non-A nucleotides (see Figure 1B in expected outcomes).The primer sequence used in this protocol is reported in Table 3.

Note:
We chose Sanger sequencing from BaseClear for our PCR products, but any type of DNA sequencing that can accurately read the poly(A)-tail will work.
CRITICAL: The presence of non-A nucleotides in the PCR products will be propagated in the mRNA molecules.8][19][20] If non-A peaks are observed, the decision of continuing with IVT of the PCR products depends on the following applications.However, if the molecules are used for kinetic and binding analysis, we suggest discarding molecules that show clear peaks associated to non-A nucleotides.

In vitro transcription of poly(A)-tailed GFP coding mRNAs
Timing: 9-10 h The tailed PCR products are in vitro transcribed to generate mRNAs with defined poly(A)-tail lengths.

Note:
The protocol describes the mRNA synthesis using the MEGAscript T7 Transcription Kit (Invitrogen), but any other T7-based transcription kit can also be used.If the T7 wild-type polymerase is exchanged with mutant polymerases, we recommend carefully checking the quality of the final mRNA products.9. Mix the reagents reported in Table 6.

Note:
Volumes are for a 20 mL reaction, but they can be scaled up if needed.
10. Incubate the reactions at 37 C for 4 h.Note: The incubation time depends on the size and transcriptional efficiency of the template, and therefore could vary.For transcripts shorter than 500 nucleotides, extending the incubation time to approximately 16 h can be beneficial.This is because a greater number of transcription initiation events are necessary to produce a specific quantity of RNA compared to longer transcripts.
11. Add 1 mL of TURBO DNase, mix well and incubate at 37 C for 15 min.12. Purify the mRNAs with following the desired protocol.
Note: Here we present two types of purification methods, you can choose the most suitable one depending on your application.
a. Phenol:Chloroform extraction followed by and isopropanol precipitation.
Note: This method is the most thorough for purifying transcripts, effectively eliminating all enzymes and most free nucleotides. i.
Bring the reaction volume to 500 mL by adding RNase-free water.Mix well.ii.
Add a mixture consisting of an equal volume of Phenol:Chloroform:Isoamyl Alcohol.Note: While this method efficiently eliminates unincorporated nucleotides and the majority of proteins, it is not ideal for precipitating RNAs smaller than 300 nucleotides.To ensure effective precipitation, the RNA concentration should be > 0.1 mg/mL.i. Add 30 mL of RNase-free water and 30 mL of 5 M LiCl (final concentration 2.5 M) and mix thoroughly.ii.Incubate samples at À20 C for at least 30 min.iii.Centrifuge samples at 4 C for 15 min and top speed.iv.Dispose of the supernatant.v. Wash the pellet with 1 mL of ice cold 70% ethanol.
CRITICAL: You should only rinse the pellet, not resuspend it.
vi. Centrifuge for 15 min at 4 C at top speed.vii.Remove the supernatant.viii.Allow the pellet to air dry.ix.Reconstitute the pellet in RNase-free water by gently pipetting up and down.
Note: If the pellet is not properly dissolving in water, you could try to heat up the samples to 37 C. CRITICAL: Try to minimize the volume used for resuspension, in order to have highly concentrated products.
13. Quantify the purified RNA using a Qubit RNA kit (High Sensitivity or Broad Range), following manufacturer's instructions.
Note: If the IVT reaction yield is high, you most likely will have to dilute your samples before quantification as their concentration will be out of range and the Qubit measurement will not be possible.We recommend performing 3 dilution replicates per sample and finally calculate their average concentration to have a reliable and accurate quantification.
Pause point: The purified IVT products can be stored at À20 C or À80 C for multiple months.Avoid freeze/thawing cycles.We suggest making aliquots of your samples to minimize these.

Timing: 8-10 h
It is recommended to check the quality of the IVT products by gel or capillary electrophoresis and sequencing.The electrophoresis analysis can highlight the presence of unspecific products and is crucial for verifying the expected size of the mRNA and of the inserted poly(A)-tails.On the other Protocol g.The bands should be of the expected size and should increase with increased poly(A)-tail length.17.If the mRNAs show the expected size and no unspecific products, you can proceed with sequencing to inspect the quality of the poly(A)-tail.
Note: The sequencing is not performed directly on the RNA, but on reverse transcribed and PCR amplified product.
a. Ligate the DNA oligonucleotide linker modified with 5 0 PO 4 and 3 0 NH 2 to the 3 0 end of the mRNAs with different poly(A)-tail lengths.b.Assemble a 25 mL ligation reaction as indicated in Table 7. c.Place the reaction mixture in a thermomixer and incubate it for 3 h at 25 C while shaking at 500 rpm.d.Quench the ligation reaction by adding 0.5 mL EDTA (0.5 M, pH 8.3).e. Aliquot 2.5-5 mL of ligated RNA products for reverse transcription.f.Reverse transcribe the ligated RNA products using a DNA primer complementary to the linker (see Table 3 for the sequence used by us).g.Assemble the reverse transcription mixture in a 20 mL reaction as indicated in Table 8.

Note:
The amount of linker-ligated RNA can be varied to improve the product quality and/or yield if desired.Generally, within these boundaries the result should be satisfactory.(see Table 3 for the primer sequences used by us).n.Incubate the reaction in a thermocycler following the conditions reported in Table 10.o.Mix the sample with 4 mL of TriTrack DNA Loading Dye (63) and load it on a 1% agarose gel, as explained in step 6.

Note:
The fragment of interest should be sharp at this point with minimal smearing or other fragments surrounding it.
p. Excise the amplified DNA products from the gel under UV light and purify them using a QIAquick gel extraction kit: i. Cut the DNA fragment from the gel with a scalpel.
ii. Purify the DNA fragment in a QIAquick Spin Column following the manufacturer's instructions.q.Sequence the PCR products using a forward primer that binds 200-300 nt upstream to the poly(A)-tail and check that the poly(A)-tail does not contain non-A nucleotides (see Figure 2C in expected outcomes).See Table 3 for the primer sequence used by us.

Note:
We chose Sanger sequencing from BaseClear for our PCR products, but any type of DNA sequencing that can accurately read the poly(A)-tail will work.

In vitro PABPCs binding assay and mobility shift assay
Timing: 2 h After having synthesized the desired poly(A)-tailed mRNA transcripts, you can proceed with the in vitro PABPCs binding and visualization through shift mobility assay.
Note: As purified proteins are notably expensive, the protocol presented here minimizes the reaction volumes (i.e., only 5 mL), allowing the visualization of very low amounts of RNA with bound PABPCs, which would not be visible on an agarose gel.
18. Proceed by assembling the reactions for the in vitro binding.a. Mix the components indicated in Table 11 in a 0.5 mL PCR tube (assemble on ice): Note: We recommend quantifying the PABP concentration before usage, as this might drastically differ from batch to batch.Furthermore, we recommend using 0.5 mL PCR tubes as they can efficiently collect the small volume used in the reaction at their bottom.
b. Mix well and spin down.c.Incubate the reactions at 37 C for 60 min in a PCR machine with a heated lid.d.In the meantime, prepare the Bioanalyzer, Agilent RNA 6000 Nano Chip as described in step 14a.Do not heat denature the samples.e.After incubation, load immediately 1 mL of the binding reactions in each well of the Bioanalyzer chip.
CRITICAL: Do not put the reactions on ice, but transfer them directly from the thermocycler to the chip.Run the Bioanalyzer chip immediately after loading.
f. Place the chip in the Bioanalyzer.g.Select the mRNA Nano assay from the Assay menu (Electrophoresis / RNA / mRNA Nano assay).

Data analysis and processing
Timing: 30 min-1 h 23.Install the required packages using ''pip'': 24. Import the required packages and the color palette of your choice:  Note: The number used in the dictionary refers to the concentration of PABP used in the experiment.This can be customized, as long as the number in the dictionary corresponds to the one in the csv file name.
27. Define the plot settings:

EXPECTED OUTCOMES
The successful synthesis of PCR products with increasing poly(A)-tail length is visible in a non-denaturing agarose gel, showing a single band for each lane, and bands shift upwards with increasing tail lengths (Figure 1A).The sequencing reactions of the tailed PCR products should show a sequence consisting of repeating A nucleotides (Figure 1B), of a length close to the expected one (i.e., the one used in the tailed primers).
The in vitro synthesized mRNAs should show a single narrow peak when analyzed via capillary electrophoresis, corresponding to the expected length of the poly(A)-tailed mRNA (Figure 2A).Similar to the PCR products, the gel-like image (or the non-denaturing agarose gel) should have a single clear band for each lane, and bands shift upwards with increasing tail lengths (Figure 2B).The sequencing of the tailed mRNA products should show a sequence consisting of only A nucleotides (Figure 2C).
The mobility shift assay should show a single band for the 0 mM PABP sample, with periodic appearance of peaks at larger sizes for increasing PABP concentrations (Figure 3A).It is essential to note that since the same concentration of RNA is loaded in each lane, the appearance of new peaks leads to a redistribution of the mRNAs across multiple bands of lower concentrations.This results in the formation of dimer bands (Figure 3B) and increased noise (highlighted by the red box in Figure 3A).Consequently, the signal-to-noise ratio might become lower as the mRNA signal decreases.For this reason, to better visualize and display the results, we suggest normalizing the peaks to the highest peak value of the sample (excluding the marker), as explained in step 26 and shown in Figure 3C.As the poly(A)-tail length increases, new peaks should appear at higher molecular weights (Figures 3C  and 3D).

LIMITATIONS
Although we have demonstrated accurate synthesis of poly(A)-tailed mRNAs within the range of 5 to 150 nucleotides, extending the poly(A)-tail length beyond 150 nucleotides may introduce non-specific products and impurities at both the DNA and mRNA levels.Additionally, the sensitivity of the mobility shift assay may vary depending on factors such as RNA quality, concentration, and experimental conditions, potentially impacting the detection of low-abundance mRNA species.While the in vitro approach provides a controlled environment for studying PABPC binding, it may not fully recapitulate the cellular context, potentially resulting in differences in binding kinetics or protein interactions.The application of the same protocol to other mRNA sequences, RBPs or biological contexts may necessitate further validation.

TROUBLESHOOTING Problem 1
The PCR products do not show clear bands for the long poly(A)-tails (related to step 6).

Potential solution
Try using an untailed linear DNA template instead of the circularized plasmid, or use a shorter tailed PCR product as template (e.g., use the 50 nt tailed PCR product as template for the 100 nt tailed PCR product).
Lowering the amount of PCR cycles could help decrease the appearance of unspecific products.When using long tailed reverse primers (>100 nt), these could be retained even after purification of the PCR products, and appear as small bands at the end of the gel.These should not affect the IVT reaction, but if preferred they can be removed by purifying the products of interest from the agarose gel.

Problem 2
The poly(A)-tail sequence contains a lot of non-A nucleotides (related to step 15

Problem 3
The IVT yield is very low (related to steps 8-13).

Potential solution
Template concentration: we suggest testing a range of template concentrations and determine which one that gives the highest yield.Incubation time: depending on the length of your template you might need to increase the incubation time of the IVT reaction.5 0 UTR length: we have noticed that increasing the sequence length upstream of the T7 promoter can help increasing transcription efficiency, probably by facilitating a better binding of the polymerase to the promoter.

Problem 4
No PCR product after the amplification of the linker-ligated mRNAs (relates to steps 15a-15n).

Potential solution
The reason why you do not have any PCR product after the amplification of the linker-ligated mRNAs could be related to an unsuccessful ligation reaction.T4 RNA ligase 1 has a certain sequence preference and does not ligate all RNA ends with the same efficiency.If this is the case, you can switch to ligation with T4 RNA ligase 2, truncated K227Q and a universal preadenylated miRNA cloning linker (NEB).Alternatively, optimization of the PCR annealing temperature, the number of PCR cycles or using a different primer sequence might result in higher and/or cleaner PCR product yield.

Problem 5
The shift mobility assay does not show clear peaks appearing (related to steps 16-18).

Potential solution
If the size of the mRNA transcript is very big (>2000 nt), the binding of the RBP might create a ribonucleoprotein (RNP) complex too big to enter the gel.One solution could be synthesizing a shorter mRNA transcript containing only the sequence of interest for the binding.Another cause could be related to the conditions used for the binding reaction.A solution to this problem can be testing different mRNA and protein concentrations to the ones suggested by us.Also varying the salt concentrations might influence protein/RNA binding.Ensure the RNA is of high purity.If the RNA exhibits non-specific peaks or broad peaks prior to the in vitro binding assay, it can be difficult to distinguish between different numbers of bound proteins.Improve the RNA purification process to achieve clearer results.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Maike M. K. Hansen (maike.hansen@ru.nl).

Technical contact
Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Carmen Grandi (carmen.grandi@ru.nl).

Protocol Materials availability
This study did not generate new materials.

8 .
Thaw and vortex the reagents: a. Keep the RNA Polymerase Enzyme Mix on ice.b.Keep the 103 Reaction Buffer at 20 C-22 C. CRITICAL: The reaction must be set up at 20 C-22 C because the spermidine in the 103 Reaction Buffer can cause the template DNA to coprecipitate if assembled on ice.Ensure you add the 103 Reaction Buffer after the water and ribonucleotides.

Figure 1 .
Figure 1.Quality control of the poly(A)-tail PCR products (A) Non-denaturing agarose gel showing the PCR products with increasing poly(A)-tail length.(B) Sanger sequencing of the PCR product containing a 50 or 100 nt long poly(A)-tail.

Figure 2 .
Figure 2. Quality control of the poly(A)-tailed mRNA products (A) Peaks obtained by capillary electrophoresis for mRNAs with increasing poly(A)-tail length.(B) Gel-like image obtained by capillary electrophoresis showing the size of the in vitro transcribed poly(A)-tailed mRNAs.(C) Sequence of 50, 100 and 150 nt long poly(A)-tailed mRNAs obtained from Sanger sequencing.

Figure 3 .
Figure 3. Mobility shift assay of the sequential PABP binding to the poly(A)-tail.(A) Gel-like images obtained by capillary electrophoresis for the in vitro binding of PABPC1 to mRNAs with 50, 100 and 150 nt long poly(A)-tails (50A 100A 150A).These tails respectively bind 3, 4 and 5 PABPC1 units.Samples for each poly(A)-tail length are from the same run.Concentrations shown are

Figure 3 .
Figure 3. Continued final PABPC1 concentrations.The contrast of single lanes is enhanced in the Agilent Bioanalyzer 2100 Expert Software to increase the signal.The red box highlights bands associated to noise or unspecific products, which become more evident as the signal-to-noise ratio decreases, due to the dilution of the mRNA throughout the multiple bands.(B) Electrophoresis peaks corresponding to single PABP units binding to 100 nt long poly(A)-tail, normalized to the marker of each lane.Zero values on the y axis correspond to the minimum value of each assay (0 mM, 2 mM, 4 mM and 6 mM PABP).(C) Electrophoresis peaks corresponding to single PABP units binding to 100 nt long poly(A)-tail, normalized to the highest peak value of each sample.Zero values on the y axis correspond to the minimum value of each assay (0 mM, 2 mM, 4 mM and 6 mM PABP).(D) Electrophoresis peaks corresponding to single PABP units binding to 50 and 150 nt long poly(A)-tail, normalized as in (C).

Table 1 .
Basic primer sequence needed for the incorporation of the T7 promoter and other regulatory sequences in the template of interest

Table 2
. Primer sequences for the introduction of the T7 promoter and poly(A)-tails of different length

Table 3 .
Primers and linker sequences for the quality control of the PCR and mRNA products Dissolve 101.65 g MgCl 2 in 400 mL of RNase-free water.Bring the final volume to 500 mL with RNase-free water.Autoclave the solution and store at RT for a maximum of 1 year.KCl in 400 mL of RNase-free water.Bring the final volume to 500 mL with RNasefree water.Autoclave the final solution and store it at RT for a maximum of 1 year.Dissolve the reagents in 200 mL of RNase-free water and add the EDTA solution.Adjust the pH to 7.0 and the final volume to 250 mL with RNase-free water.Store the final solution 20 C-22 C protected from light for a maximum of 6 months.
Dissolve the reagents in 800 mL of RNase-free water.Adjust the final volume to 1 L with RNase-free water.Store at RT for a maximum of 1 year.If the buffer becomes cloudy or discolored, discontinue use and discard.
Mix the solutions and store the buffer at RT for maximum 1 year.

Table 4 .
Reagents for a single 100 mL PCR reaction for the introduction of the poly(A)-tails

Table 5 .
PCR program used for the synthesis of tailed PCR products CRITICAL: Phenol:Chloroform:Isoamyl Alcohol is toxic if inhaled, and can cause eye damage and severe skin burns.Prolonged exposure can damage organs.When handling, work in a fume hood and wear protective goggles.Transfer again the aqueous phase into a clean tube.xii.Add 1 volume of isopropanol and mix it by reversing 5-6 times.xiii.Incubate at À20 C for at least 30 min.xiv.Centrifuge for 15 min at 4 C, 12000 g. xv.Dispose of the supernatant.

Table 6 .
Reagents for a single 20 mL IVT reaction

Table 7 .
Reagents for a single 25 mL ligation reaction

Table 8 .
Reagents for a single reverse transcription reaction of the linker-ligated products

Table 9 .
Reagents for a 20 mL PCR reaction for the amplification of the reverse transcribed cDNA products

Table 10 .
PCR program used for the amplification of the cDNA products Protocol 19.After running, visualize the data as explained in step 14a.20.Export raw data to further process and normalize.To export the csv files: a. Click on File / Export.The ''Electrophoresis Export Option'' window will open.b.Check the ''Result Tables'' to export a csv file containing all result table values (make sure to include the ladder).c.Check the ''Sample Data'' tab to export one file per sample.d.Check the ''Gel Image'' tab to export the gel like image (see Figure 3A in expected outcomes for some examples).21.The raw data contained in the csv files exported from the Agilent Bioanalyzer 2100 Expert Software can be processed and plotted in Python.Here we show how to normalize the Bioanalyzer peaks in two ways: a.To the marker peak.b.To the highest peak of each sample.22.To do this, you will need the following files: a.One csv file containing the Ladder information.Note: When you export the ''Result Table'' csv file, make sure to check the tab ''Include Ladder''.You can then just copy the Ladder peak table in a new csv file (for an example see the provided file 'Ladder_peaks.csv').
Note:The number of cycles can be varied to improve the product quality and/or yield if desired.Generally, within these boundaries the result should be satisfactory.STAR Protocols 5, 103284, September 20, 2024

Table 11 .
Reagents for a 5 mL PABP binding reaction to poly(A)-tailed mRNAs ). STAR Protocols 5, 103284, September 20, 2024 Protocol Potential solution DNA polymerase: if the problem appears to be on the PCR product level, try testing different DNA polymerases for template amplification, as fidelity varies among different polymerases.RNA polymerase: similar to the DNA polymerases, different RNA polymerases can have different fidelities.Therefore, testing different mutants or types could help obtain the desired products.