Routh A, Ji P, Jaworski E, Xia Z, Li W, Wagner EJ (2017). Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation..
Elrod ND, Jaworski EA, Ji P, Wagner EJ, Routh A (2019). Development of Poly(A)-ClickSeq as a tool enabling simultaneous genome-wide poly(A)-site identification and differential expression analysis..
Routh A, Head SR, Ordoukhanian P, Johnson JE (2015). ClickSeq: Fragmentation-Free Next-Generation Sequencing via Click Ligation of Adaptors to Stochastically Terminated 3'-Azido cDNAs..
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
ClickSeq‱ is a pending trademark of ClickSeq Technologies, LLC.
Abstract
Poly(A)-ClickSeq is a library preparation method used to target the 3’ ends of polyadenylated RNA, such as eukaryotic mRNAs. This technique offers an alternative to conventional RNA-seq methods that provide the user with sequencing reads that cover entire transcripts. Instead, the 3’ end targeting protocol of Poly(A)-ClickSeq enables a more cost efficient and straightforward method for measuring differential gene expression and simultaneously the mapping of poly(A) sites which can be used for alternative polyadenylation studies.
The process takes advantage of the chain-terminating properties of 3′-azido-nucleotides, which are included the initial in vitro reverse-transcription reactions uniformly required for RNAseq. In Poly(A)-ClickSeq (PAC-Seq), priming occurs from poly(A)-tails using an unanchored oligo-dT primer and only AzATP, AzGTP and AzCTP (collectively known as AzVTPs) are supplemented in the RT reaction. As a result, cDNA synthesis does not terminate in the poly(A)-tail, but rather continues until the 3’UTR is reached. Thereafter, the modified nucleotides (AzVTPs) are stochastically incorporated into the nascent cDNA at a programmable distance upstream of the 3’UTR/Poly(A)-tail junction, yielding cDNA fragments blocked at their 3′ends with azido groups. The 3′-azido-blocked cDNA fragments are ‘click-ligated’ onto alkyne-functionalized sequencing adaptors, which can subsequently be PCR-amplified to yield a sequencing-ready NGS library.
PAC-Seq offers unique advantages over common RNA sequencing and 3’end mapping protocols in that it does not require the purification, selection, or fragmentation steps typically required in RNA-seq approaches. Sample preparation is started directly from crude total cellular RNA. Furthermore, click-chemistry is utilized to attach the required sequencing adapter, rather than commonly-used enzymatic reactions. Overall, this results in increased efficiency of the protocol, fewer processing steps, and reduced time from RNA to sequencing-ready libraries.
Guidelines
Next-Generation Sequencing (NGS) is a highly sensitive technique that generates millions of data points. The quality of your input material can be translated to the final quality of your libraries at the end of
this protocol, and in turn, the sequencing data. Use common laboratory precautions to minimize introducing contamination to your samples and follow procedures as written to ensure good yields.
RNA Handling
Work in an RNase-free environment; use RNase inhibitors to decontaminate your workspace. Follow standard aseptic techniques.
Wear PPE (gloves, lab coat, eye protection, etc) to protect your workstation and reagents from RNases that are present on your skin. Change gloves often.
Use RNase-free plasticware by purchasing certified materials or by treating consumables with RNase Inhibitors.
RNA Input Guidelines
Most standard RNA extraction protocols are compatible with this method. Take care during the final steps of the extraction method to ensure that no salts, metal ions, or organic solvents are carried over into the final elution step. For example, ethanol contamination can reduce the efficiency of all reactions in this protocol.
During the RNA extraction elute your sample in RNase-free water or Tris buffer (10mM, pH 7.4). Do NOT use carrier RNA during the extraction process. Commonly, carrier RNA is poly(A) oligos. Do not use these, they will negatively impact your final sequencing data as you will only sequence this RNA
We recommend using protocols that do not co-purify genomic DNA, since A-rich genomic DNA may also be captured by the Poly(A)-ClickSeq approach. If available, complete the specified DNase I treatment during RNA extraction.
RNA can be quantified by any of the user’s preferred methods (UV-vis, spectrophotometer, Qubit fluorometer, etc.)
This protocol has been demonstrated to work with as little as 30ng up to 4µg of total cellular RNA. That quantity should be in a max of 10µl water or Tris- buffer. While it is possible to use the specified range of starting material, we have found that the optimal amount to start with is generally 1µg of total cellular RNA.
A260/A280 values should be between 1.9 and 2.2
RIN values should be >6.0
Bead Handling
Follow manufacturers recommendations but generally, SPRI purification beads should be stored at +4°C. Beads tend to settle during storage so they should be resuspended thoroughly before use (by vortexing or pipetting vigorously). Beads are resuspended properly when the solution is uniform in color (light brown) and there are no visible clumps on the bottom or sides of the tube.
SPRI beads are magnetic and are collected by placing the sample tube on a magnetic rack. The time it takes for the beads to pellet will depend on the strength of the magnet you are using; adjust the incubation time accordingly by waiting until the solution is completely clear. Waiting longer to ensure that all the beads have pelleted will not affect overall quality of your libraries but will ensure adequate efficiency of the purification steps.
When discarding the supernatant of pelleted beads, take care to not disturb the beads by keeping the sample tube on the magnetic rack and do not touch the pellet with a pipette tip.
Ethanol carryover after the second wash step during bead purification can inhibit subsequent reactions. Visually inspect each well to ensure all ethanol has been removed.
Do not allow beads to over-dry, exhibited by visible cracking. This can damage the beads and reduce overall yields.
General
Read an entire section of the protocol before beginning to familiarize yourself with all steps. To minimize any issues, collect the necessary equipment, prepare the appropriate reagents, and pre-load the appropriate incubation temperatures on your thermocycler.
Enzymes should be thawed and kept on ice while in use. All other reagents can be thawed at room temperature and kept on ice while not in use. SPRI beads equilibrate to room temperature prior to use.
Spin down all reagent tubes prior to opening to prevent loss and to minimize cross-contamination.
Use calibrated pipettes and fresh tips between samples and reagents.
Pipette reagents and mixes carefully and in a controlled manner. Viscous reagents (such as enzyme mixes) should be pipetted slowly to ensure accuracy and the complete transfer of the reagent. Avoid frothing and the introduction of air bubbles while mixing.
Master Mixes
Steps #4 and #17 require the generation of master mixes. In order to have enough solution for
all samples, include a 10% surplus per reaction when calculating the master mix.
PCR Cycle Optimization
The number of PCR cycles to perform will depend on the sample type (species, tissue, quality, etc.) so optimizations should be completed prior to processing all samples of the same type. This protocol has been extensively tested using total cellular RNA extracted from D. melanogaster (S2) cells. The provided values should be used as a reference only.
A
B
Total RNA Input
PCR Cycle Number
100ng
17-19
500ng
16-18
1µg
13-15
2µg
12-14
Sequencing Guidelines
Final Poly(A)-ClickSeq libraries are compatible with the Illumina sequencing platforms (NextSeq, NovaSeq, MiSeq, HiSeq, etc.) or with Element Biosciences Aviti™ systems/flowcells that are compatible with the same Illumina adaptors.
Final ClickSeq libraries can also be sequenced on Oxford Nanopore Technology's flowcell by straight-forward ligation of the ONT DNA sequencing (e.g. LSK109) adaptors.
Read 1 will include 4nt of the UMI followed by the cDNA fragment.
The 8nt i7 index sequences are provided in the table below.
A
B
Index
Sequence
D701
ATTACTCG
D702
TCCGGAGA
D703
CGCTCATT
D704
GAGATTCC
D705
ATTCAGAA
D706
GAATTCGT
D707
CTGAAGCT
D708
TAATGCGC
D709
CGGCTATG
D710
TCCGCGAA
D711
TCTCGCGC
D712
AGCGATAG
Materials
Required Reagents
Poly(A)-ClickSeq KitClickSeq Technologies LLC
SuperScript™ III Reverse TranscriptaseThermo FisherCatalog #18080093
OneTaq 2X Master Mix with Standard Buffer - 100 rxnsNew England BiolabsCatalog #M0482S
SPRIselect reagent kitBeckman CoulterCatalog #B23317 or equivalent DNA/RNA Purification Beads
OneTaq 2X Master Mix with Standard Buffer - 100 rxnsNew England BiolabsCatalog #M0482S
Materials, Step 26
RNase H - 250 unitsNew England BiolabsCatalog #M0297S
Materials, Step 7
Safety warnings
Standard molecular lab precautions should be adhered to, including standard PPE (gloves, lab coat, eye protection, etc).
Before start
Check to ensure that you have all the necessary components, materials, and equipment before beginning this protocol. The protocol can be grouped into 6 broad steps/sections:
Reverse Transcription and RNA Removal: Total RNA is reverse transcribed by priming from the poly(A) tail of mRNA and other transcripts. The presence of AzVTPs stochastically terminates the reaction upstream of the 3’UTR/Poly(A)-tail junction, generating a distribution of randomly sized cDNA fragments.
First Bead Purification: Magnetic beads are used to remove all components of the reverse transcription reaction leaving the cDNA fragments for further processing.
Click-Ligation: During this step, a sequencing adapter is attached to the azido-terminated 3’ ends of the cDNA fragments using a Click-Chemistry reaction.
Second Bead Purification: SPRI beads are used to remove components of the click-ligation reaction leaving cDNA fragments that are flanked by sequencing adapters.
PCR Library Amplification: At this step, PCR is used to convert the single stranded cDNA fragments to dsDNA fragments, amplify the fragments to generate enough material for sequencing, and to add the sequencing indices/barcodes (Illumina i7 adapters).
Final Bead Purification: Magnetic bead purification is used to remove components of the PCR amplification reaction from the completed barcoded libraries and return size-selected (~200-400bp) sequencing-ready libraries. If you are using this protocol for alternative polyadenylation studies, please reference Appendix C for an additional size selection step.
Poly(A)-ClickSeq Schematic
Reverse Transcription and RNA Removal
Reverse Transcription and RNA Removal
50m
50m
In a 0.2ml tube, dilute 100 ng-2 µg of input RNA to 10 µL using nuclease free water.
Note
This protocol has been demonstrated to work with as little as 30ng up to 4µg of total cellular RNA. That quantity should be in a max of 10µl water or Tris-buffer. While it is possible to use the specified range of starting material, we have found that the optimal amount to start with is generally 1µg of total cellular RNA. Below this value, additional PCR cycles are required that will result in PCR duplication and increased adaptor-dimers in the final library.
Total cellular RNA can be used as the input material. There is no need to poly(A)-select or ribo-deplete your samples, since the Poly(A)-ClickSeq process primes from the poly(A)-tail of mRNA transcripts.
Add 3 µL of PAC Primer Mix (PPM) to the diluted RNA. Mix well.
Incubate the mixture at 65 °C for 00:05:00 to melt any RNA secondary structure and immediately snap cool the reaction by placing the tubes on ice for 00:01:00 to anneal the reverse primer.
Note
Alternative annealing conditions may improve the yield and on-target priming of the reverse-transcription reaction. For example, incubating the sample at 65 °C for 00:05:00 may be followed by slow cooling by placing on tube racks at room temperature.
6m
After snap cooling, generate an RT master mix in a separate tube by combining the following components, pipette well to mix:
Generate a Master Mix of these components when preparing more than one sample at a time
Note
Recombinant Ribonuclease Inhibitor is not essential in cases where RNA quality or abundance is not a concern and can be replaced with nuclease-free water to save on reagent costs
Add 7 µL of the RT master mix to each reaction and pipette to mix.
Incubate the reaction in a thermocycler using the following conditions:
25 °C for 00:10:00
50 °C for 00:10:00
75 °C for 00:10:00
4 °C for ∞
Note
Skipping the 25 °C incubation step may improve specificity/on-target priming.
At this point it is recommended to remove the SPRI Bead reagents from 4°C storage to allow them to equilibrate to room temperature.
30m
[optional] To remove template RNA, add 0.5 µL of RNase H - 250 unitsNew England BiolabsCatalog #M0297S. Pipette to mix. Incubate the reaction in a thermocycler using the following conditions:
37 °C for 00:20:00
80 °Cfor 00:10:00
4 °C for ∞
Note
Removal of the RNA template using RNaseH is not essential but may improve library yield in some cases. In cases where library yield is not a concern, step #7 may be omitted to save on reagent costs and prep time.
30m
First Bead Purification
First Bead Purification
15m
15m
Add 36 µL of thoroughly resuspended SPRIselect reagent kitBeckman CoulterCatalog #B23317 beads to the reaction mix. Mix well by pipetting. Incubate for 00:05:00 at Room temperature.
Note
SPRI beads tend to settle during storage and should bethoroughly resuspended by vortexing briefly prior to use. Additionally, it is important to allow the SPRI beads to equilibrate to room temperature for 30 min.
5m
Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 00:05:00 or until the supernatant is completely clear.
5m
Leaving the sample tubes on the magnetic rack, discard clear supernatant taking care to not disturb the pelleted beads.
Leaving the sample tubes on the magnetic rack, wash pelleted beads by adding 200 µLof freshly prepared 80% EtOH. Do not resuspend beads. After 00:00:30 incubation remove and discard the supernatant.
30s
Repeat the EtOH washing step (#11) for a total of two washes. After the second wash make sure to remove all traces of EtOH as ethanol can impair the efficiency of subsequent steps. Visually inspect tubes for trace amounts of EtOH left over on the sides of the tubes. Tubes should be removed off the magnet and pulse spun to collect extra EtOH at the bottom of the sample tube. Place tubes back on a magnetic stand and pipette off any remaining EtOH.
Remove the sample tubes from the magnetic rack and resuspend the beads by adding 21 µL of Elution Buffer 1 (EB1).Incubate resuspended beads for 00:02:00 at Room temperature
2m
Place the sample tubes back on the magnetic rack and allow beads to pellet. Transfer 20 µL of the supernatant to a fresh 0.2ml sample tube.
Safe stopping point: samples can be stored at -20 °C
Click-Ligation
Click-Ligation
20m
20m
Add 15 µL of Click Mix (CM) to each sample. Pipette to mix, taking care to not introduce air bubbles.
Note
The Click-Adaptor present in the Click Mix (CM) is:
In a separate tube, prepare the Click Ligation master mix, pipetting up and down 3-5 times to mix, taking care to not introduce any air bubbles. CC is blue in color and should turn clear/colorless when mixed properly. This is a time sensitive reaction so proceed immediately to the next step.
A
B
C
Per Rxn
Master Mix
Click Accelerant (CA)
4µl
Click Catalyst (CC)
1µl
Generate a Master Mix of these components when preparing more than one sample at a time
Note
The tube containing CA should only be used one time to limit exposure to atmospheric oxygen. Discard the tube once it has been used. The ClickSeq kit provides two CA tubes.
Add 5 µL of the Click Ligation master mix to each sample tube. Once the mix has been added to all sample tubes, pipette or flick to mix, and spin down contents of the tube. Incubate the reaction at room temperature for 00:15:00.
15m
Second Bead Purification
Second Bead Purification
15m
15m
Add 64 µL of thoroughly resuspended SPRIselect reagent kitBeckman CoulterCatalog #B23317beadsto the reaction mix. Mix well by pipetting. Incubate for 00:05:00 at Room temperature
5m
Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 00:05:00or until the supernatant is completely clear.
5m
Leaving the sample tubes on the magnetic rack, discard clear supernatant. Take care to not disturb the pelleted beads.
Leaving the sample tubes on the magnetic rack, wash pelleted beads by adding 200 µL of freshly prepared 80% EtOH. Do not resuspend beads. After 00:00:30 incubation remove and discard the supernatant.
30s
Repeat the EtOH wash step (#21) for a total of two washes. After the second wash make sure to remove all traces of EtOH as ethanol can impair the efficiency of subsequent steps. Visually inspect tubes for trace amounts of EtOH left over on the sides of the tubes. Tubes should be removed off the magnet and pulse spun to collect extra EtOH at the bottom of the tube. Place tubes back on a magnetic stand and pipette off any remaining EtOH.
Remove the sample tubes off the magnetic rack and resuspend the beads by adding 21 µLof Elution Buffer 2 (EB2).Incubate resuspended beads for 00:02:00 at Room temperature.
2m
Place the sample tubes back on the magnetic plate and allow beads to pellet. Transfer 20 µL of the supernatant to a fresh 0.2ml PCR tube.
Safe stopping point: samples can be stored at -20 °C overnight
PCR Library Amplification
PCR Library Amplification
2h 10m
2h 10m
Transfer 10 µL of the sample volume to a new PCR tube.
(Note: Retain the other 10µl of your sample. It can be used to repeat the PCR amplification step in the case of over- or under-cycling or for technical replicates).
Add 25 µL of OneTaq 2X Master Mix with Standard Buffer - 100 rxnsNew England BiolabsCatalog #M0482S to each sample tube.
Using a unique Index Primer per sample, add 15 µL of each respective i7 index primer (Index Primer N001-N012) to each sample tube. Pipette to mix. Take note of which index was used for each sample.
where 'xxxxxx' denotes the barcode used. Any indexing primers that conform to this 'structure' can be used here.
The 8nt i7 index sequences are provided in the table below:
A
B
D701
ATTACTCG
D702
TCCGGAGA
D703
CGCTCATT
D704
GAGATTCC
D705
ATTCAGAA
D706
GAATTCGT
D707
CTGAAGCT
D708
TAATGCGC
D709
CGGCTATG
D710
TCCGCGAA
D711
TCTCGCGC
D712
AGCGATAG
i7 Indexes included in the ClickSeq Technologies Primer Box (12 Indexes)
Place the sample tubes in a thermocycler using the following PCR cycling program:
94 °C00:01:00; 53 °C00:00:30; 68 °C00:10:00;
[94 °C00:00:30; 53 °C00:00:30; 68 °C00:02:00] x 12-21 cycles;
68 °C00:05:00;
4 °C f∞
Note
The number of PCR cycles to perform will depend on the sample type (species, tissue, quality, etc.) so optimizations should be completed prior to processing all samples of the same type. This protocol has been extensively tested using total cellular RNA extracted from D. melanogaster (S2) cells. The provided values should be used as a reference only. We recommended starting with 18 cycles when generating libraries from >100ng of high quality RNA and adjusting as needed.
A
B
Total RNA Input
PCR Cycle Number
100ng
17-19+
500ng
16-18
1µg
13-15
2µg
12-14
Suggested PCR cycle numbers depending upon the amount of RNA provided in Step #1.
Note
In the case of over- or under-cycling, starting at step #25, the protocol may be repeated using the retained 10µl of your sample from step #24. Under-cycling will result in low yield and the PCR amplification should be repeated with a higher cycle number. Over-cycling can result in excessive PCR duplication and will result in excess library.
19m 30s
Final Bead Purification
Final Bead Purification
15m
15m
Add 30 µL of thoroughly resuspended SPRIselect reagent kitBeckman CoulterCatalog #B23317 to the reaction mix. Mix well by pipetting. Incubate for 5 min at room temperature.
SPRIselect reagent kitBeckman CoulterCatalog #B23317 Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 00:05:00 or until the supernatant is completely clear.
5m
Leaving the sample tubes on the magnetic rack, transfer the supernatant to fresh 0.2ml tubes. Take care to not disturb the pelleted beads. Pelleted beads may be discarded. Do NOT discard the supernatant.
Add 15 µL of thoroughly resuspended SPRI beadsto the retained supernatant from step #31. Mix well by pipetting. Incubate for 00:05:00 at Room temperature.
5m
Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 00:05:00 or until the supernatant is completely clear.
5m
Leaving the sample tubes on the magnetic rack, discard clear supernatant. Take care to not disturb the pelleted beads.
Leaving the sample tubes on the magnetic rack, wash pelleted beads by adding 200 µL of freshly prepared 80% EtOH. Do not resuspend beads. After 00:00:30 incubation remove and discard the supernatant.
30s
Repeat the EtOH wash step (#35) for a total of two washes. After the second wash make sure to remove all traces of EtOH as ethanol can impair the efficiency of subsequent steps. Visually inspect tubes for trace amounts of EtOH left over on the sides of the tubes. Tubes should be removed off the magnet and pulse spun to collect extra EtOH at the bottom of the tube. Place tubes back on a magnetic stand and pipette off any remaining EtOH.
Remove the sample tubes off the magnetic rack and resuspend the beads by adding 18 µL of Elution Buffer 2 (EB2).Incubate resuspended beads for 00:02:00 at Room temperature.
2m
Place the sample tubes back on the magnetic plate and allow beads to pellet. Transfer 17 µL of the supernatant to a fresh tube.
Samples are now ready for quality control, quantification, pooling, and sequencing.
Safe stopping point: samples can be stored at -20 °C
Note
Following the protocol as directed will retain fragments ~200-400bp. This is the optimal size fragment size range for the Poly(A)-ClickSeq protocol for most purposes. The final fragments consist of: ~140nt of sequencing adapters, ≥21nt of A's, and ~40-240nt of the cDNA fragment. The ‘R1’ forward sequencing read will be derived from the p5 Illumina adaptor (click-ligated in step #17), and read through the UMI the cDNA fragment, and finally into the poly(A)-tail if the cDNA fragment is short than the number of sequencing cycles. Since the oligo-dT primer used in the reverse transcription is not ‘anchored’ to the junction of the 3’end and poly(A)-tail or a given mRNA, the number of A’s found in the R1 read can (and should) exceed the length of the oligo-dT primer. Indeed, this feature allows for the computational differentiation between ‘real’ poly(A)-tails and aberrant poly(A)-tails that are the result of priming internally within an mRNA transcript. Furthermore, for alternative polyadenylation analyses it is critical to capture the poly(A) sequence. Therefore, we have found that it is beneficial to do an additional size selection step to ensure that the cDNAs are uniformly short enough that the majority of ‘R1’ forward sequencing reads reach the poly(A)-tail.
[Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
[Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
Quantify samples using the Qubit dsDNA High Sensitivity Kit or with a BioAnalyzer High Sensitivity DNA kit.
Make an equimolar pool(s) of your samples (or however you would like to distribute the pool). You may pool all your samples into one pool, or you may make a few pools with fewer samples per pool. When pooling, consider the capacity of your gel electrophoresis system.
Following the protocol for the agarose system of your choosing; assemble your gel electrophoresis system, mix your sample with loading dye (if necessary), load your samples and ladder into the wells of your gel, and run the gel to separate your samples.
Using a clean gel knife and referencing the DNA ladder, excise a gel fragment between 200 and 400bp (or as required per the user’s assay).
Following the user defined agarose DNA extraction protocol, dissolve and extract the DNA from the excised agarose. Elute/resuspend your sample in 10-20µl of Elution Buffer 2 (EB2).
Samples are now ready for quality control, quantification, pooling, and sequencing.
Safe stopping point: samples can be stored at -20 °C
Protocol references
CITATION
Routh A, Ji P, Jaworski E, Xia Z, Li W, Wagner EJ (2017). Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation..
Mukherjee S, Graber JH, Moore CL (2023). Macrophage differentiation is marked by increased abundance of the mRNA 3' end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64..
Jonnakuti VS, Ji P, Gao Y, Lin A, Chu Y, Elrod N, Huang KL, Li W, Yalamanchili HK, Wagner EJ (2023). NUDT21 alters glioma migration through differential alternative polyadenylation of LAMC1..
de Prisco N, Ford C, Elrod ND, Lee W, Tang LC, Huang KL, Lin A, Ji P, Jonnakuti VS, Boyle L, Cabaj M, Botta S, Õunap K, Reinson K, Wojcik MH, Rosenfeld JA, Bi W, Tveten K, Prescott T, Gerstner T, Schroeder A, Fong CT, George-Abraham JK, Buchanan CA, Hanson-Khan A, Bernstein JA, Nella AA, Chung WK, Brandt V, Jovanovic M, Targoff KL, Yalamanchili HK, Wagner EJ, Gennarino VA (2023). Alternative polyadenylation alters protein dosage by switching between intronic and 3'UTR sites..
Vu MN, Lokugamage KG, Plante JA, Scharton D, Bailey AO, Sotcheff S, Swetnam DM, Johnson BA, Schindewolf C, Alvarado RE, Crocquet-Valdes PA, Debbink K, Weaver SC, Walker DH, Russell WK, Routh AL, Plante KS, Menachery VD (2022). QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis..
Sotcheff SL, Chen JY, Elrod N, Cao J, Jaworski E, Kuyumcu-Martinez MN, Shi PY, Routh AL (2022). Zika Virus Infection Alters Gene Expression and Poly-Adenylation Patterns in Placental Cells..
LaForce GR, Farr JS, Liu J, Akesson C, Gumus E, Pinkard O, Miranda HC, Johnson K, Sweet TJ, Ji P, Lin A, Coller J, Philippidou P, Wagner EJ, Schaffer AE (2022). Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration..
Scarborough AM, Flaherty JN, Hunter OV, Liu K, Kumar A, Xing C, Tu BP, Conrad NK (2021). SAM homeostasis is regulated by CFI(m)-mediated splicing of MAT2A..
Montalbano M, Jaworski E, Garcia S, Ellsworth A, McAllen S, Routh A, Kayed R (2021). Tau Modulates mRNA Transcription, Alternative Polyadenylation Profiles of hnRNPs, Chromatin Remodeling and Spliceosome Complexes..
Li L, Huang KL, Gao Y, Cui Y, Wang G, Elrod ND, Li Y, Chen YE, Ji P, Peng F, Russell WK, Wagner EJ, Li W (2021). An atlas of alternative polyadenylation quantitative trait loci contributing to complex trait and disease heritability..
Enwerem III, Elrod ND, Chang CT, Lin A, Ji P, Bohn JA, Levdansky Y, Wagner EJ, Valkov E, Goldstrohm AC (2021). Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome..
Cao J, Verma SK, Jaworski E, Mohan S, Nagasawa CK, Rayavara K, Sooter A, Miller SN, Holcomb RJ, Powell MJ, Ji P, Elrod ND, Yildirim E, Wagner EJ, Popov V, Garg NJ, Routh AL, Kuyumcu-Martinez MN (2021). RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts..
Alcott CE, Yalamanchili HK, Ji P, van der Heijden ME, Saltzman A, Elrod N, Lin A, Leng M, Bhatt B, Hao S, Wang Q, Saliba A, Tang J, Malovannaya A, Wagner EJ, Liu Z, Zoghbi HY (2020). Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation..
Chu Y, Elrod N, Wang C, Li L, Chen T, Routh A, Xia Z, Li W, Wagner EJ, Ji P (2019). Nudt21 regulates the alternative polyadenylation of Pak1 and is predictive in the prognosis of glioblastoma patients..
Elrod ND, Jaworski EA, Ji P, Wagner EJ, Routh A (2019). Development of Poly(A)-ClickSeq as a tool enabling simultaneous genome-wide poly(A)-site identification and differential expression analysis..
Routh A, Ji P, Jaworski E, Xia Z, Li W, Wagner EJ. Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation.
Elrod ND, Jaworski EA, Ji P, Wagner EJ, Routh A. Development of Poly(A)-ClickSeq as a tool enabling simultaneous genome-wide poly(A)-site identification and differential expression analysis.
de Prisco N, Ford C, Elrod ND, Lee W, Tang LC, Huang KL, Lin A, Ji P, Jonnakuti VS, Boyle L, Cabaj M, Botta S, Õunap K, Reinson K, Wojcik MH, Rosenfeld JA, Bi W, Tveten K, Prescott T, Gerstner T, Schroeder A, Fong CT, George-Abraham JK, Buchanan CA, Hanson-Khan A, Bernstein JA, Nella AA, Chung WK, Brandt V, Jovanovic M, Targoff KL, Yalamanchili HK, Wagner EJ, Gennarino VA. Alternative polyadenylation alters protein dosage by switching between intronic and 3'UTR sites.
Vu MN, Lokugamage KG, Plante JA, Scharton D, Bailey AO, Sotcheff S, Swetnam DM, Johnson BA, Schindewolf C, Alvarado RE, Crocquet-Valdes PA, Debbink K, Weaver SC, Walker DH, Russell WK, Routh AL, Plante KS, Menachery VD. QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis.
Montalbano M, Jaworski E, Garcia S, Ellsworth A, McAllen S, Routh A, Kayed R. Tau Modulates mRNA Transcription, Alternative Polyadenylation Profiles of hnRNPs, Chromatin Remodeling and Spliceosome Complexes.
Mukherjee S, Graber JH, Moore CL. Macrophage differentiation is marked by increased abundance of the mRNA 3' end processing machinery, altered poly(A) site usage, and sensitivity to the level of CstF64.
Cao J, Verma SK, Jaworski E, Mohan S, Nagasawa CK, Rayavara K, Sooter A, Miller SN, Holcomb RJ, Powell MJ, Ji P, Elrod ND, Yildirim E, Wagner EJ, Popov V, Garg NJ, Routh AL, Kuyumcu-Martinez MN. RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts.
Chu Y, Elrod N, Wang C, Li L, Chen T, Routh A, Xia Z, Li W, Wagner EJ, Ji P. Nudt21 regulates the alternative polyadenylation of Pak1 and is predictive in the prognosis of glioblastoma patients.
LaForce GR, Farr JS, Liu J, Akesson C, Gumus E, Pinkard O, Miranda HC, Johnson K, Sweet TJ, Ji P, Lin A, Coller J, Philippidou P, Wagner EJ, Schaffer AE. Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration.
Routh A, Ji P, Jaworski E, Xia Z, Li W, Wagner EJ. Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation.
Sotcheff SL, Chen JY, Elrod N, Cao J, Jaworski E, Kuyumcu-Martinez MN, Shi PY, Routh AL. Zika Virus Infection Alters Gene Expression and Poly-Adenylation Patterns in Placental Cells.
Li L, Huang KL, Gao Y, Cui Y, Wang G, Elrod ND, Li Y, Chen YE, Ji P, Peng F, Russell WK, Wagner EJ, Li W. An atlas of alternative polyadenylation quantitative trait loci contributing to complex trait and disease heritability.
Enwerem III, Elrod ND, Chang CT, Lin A, Ji P, Bohn JA, Levdansky Y, Wagner EJ, Valkov E, Goldstrohm AC. Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome.
Alcott CE, Yalamanchili HK, Ji P, van der Heijden ME, Saltzman A, Elrod N, Lin A, Leng M, Bhatt B, Hao S, Wang Q, Saliba A, Tang J, Malovannaya A, Wagner EJ, Liu Z, Zoghbi HY. Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation.
Jonnakuti VS, Ji P, Gao Y, Lin A, Chu Y, Elrod N, Huang KL, Li W, Yalamanchili HK, Wagner EJ. NUDT21 alters glioma migration through differential alternative polyadenylation of LAMC1.
