TITLE: Poly(A)-ClickSeq: Poly(A)-Primed Protocol with Single Indexing using Poly(A)-ClickSeq Kit
AUTHORS: Andrew Routh, Elizabeth Jaworski

[DESCRIPTION]
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. 

[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. 


 



[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.

 


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. 

 



[STEPS]
SECTION: Reverse Transcription and RNA Removal
1. In a 0.2ml tube, dilute 100 ng-2 µg of input RNA to 10 µL using nuclease free water.

 

SECTION: Reverse Transcription and RNA Removal
2. Add 3 µL of PAC Primer Mix (PPM) to the diluted RNA. Mix well.

 

SECTION: Reverse Transcription and RNA Removal
3. Incubate the mixture at 65 °C for 5 min to melt any RNA secondary structure and immediately snap cool the reaction by placing the tubes on ice for 1 min to anneal the reverse primer.
 


SECTION: Reverse Transcription and RNA Removal
5. Add 7 µL of the RT master mix to each reaction and pipette to mix.

SECTION: Reverse Transcription and RNA Removal
6. Incubate the reaction in a thermocycler using the following conditions:
 25 °C for 10 min
 50 °C for 10 min
 75 °C for 10 min
 4 °C for ∞

 

SECTION: Reverse Transcription and RNA Removal
7. [optional] To remove template RNA, add 0.5 µL of  . Pipette to mix. Incubate the reaction in a thermocycler using the following conditions:  
37 °C for 20 min
80 °Cfor 10 min
4 °C for ∞

 

SECTION: First Bead Purification
8. Add 36 µL of thoroughly resuspended   beads to the reaction mix. Mix well by pipetting. Incubate for 5 min at Room temperature.

 

SECTION: First Bead Purification
9. Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 5 min or until the supernatant is completely clear.

SECTION: First Bead Purification
10. Leaving the sample tubes on the magnetic rack, discard clear supernatant taking care to not disturb the pelleted beads.
SECTION: First Bead Purification
11. Leaving the sample tubes on the magnetic rack, wash pelleted beads by adding 200 µLof freshly prepared 80% EtOH. Do not resuspend beads. After 30 s incubation remove and discard the supernatant.

SECTION: First Bead Purification
12. 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. 
SECTION: First Bead Purification
14. 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

SECTION: Click-Ligation
15. Add 15 µL of Click Mix (CM) to each sample. Pipette to mix, taking care to not introduce air bubbles.

 

SECTION: Click-Ligation
16. 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.

       Per Rxn   Master Mix     Click Accelerant (CA   )   4µl         Click Catalyst (CC   )   1µl       

 

SECTION: Click-Ligation
17. 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 15 min.

SECTION: Reverse Transcription and RNA Removal
4. After snap cooling, generate an RT master mix in a separate tube by combining the following components, pipette well to mix:
  
       Per Rxn   Master Mix     5X First Strand Buffer   4µl         DTT (100mM)   1µl         [optional] RNaseOUT™ Recombinant Ribonuclease Inhibitor [or replace with nuclease-free water]   1µl         SSIII Reverse Transcriptase™   1µl       

 

SECTION: Second Bead Purification
18. Add 64 µL of thoroughly resuspended  beads to the reaction mix. Mix well by pipetting. Incubate for 5 min at Room temperature
SECTION: Second Bead Purification
19. Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 5 minor until the supernatant is completely clear.
SECTION: Second Bead Purification
20. Leaving the sample tubes on the magnetic rack, discard clear supernatant. Take care to not disturb the pelleted beads.
SECTION: Second Bead Purification
21. 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 30 s incubation remove and discard the supernatant.
SECTION: Second Bead Purification
22. 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. 
SECTION: Second Bead Purification
23. Remove the sample tubes off the magnetic rack and resuspend the beads by adding 21 µLof Elution Buffer 2 (EB2). Incubate resuspended beads for 2 min at Room temperature.
SECTION: Second Bead Purification
24. 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
SECTION: PCR Library Amplification
25. 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).
SECTION: PCR Library Amplification
26. Add 25 µL of   to each sample tube.

SECTION: PCR Library Amplification
27. 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. 

 
 

SECTION: PCR Library Amplification
28. Place the sample tubes in a thermocycler using the following PCR cycling program:
94 °C 1 min; 53 °C 30 s; 68 °C 10 min; 
[94 °C 30 s; 53 °C30 s; 68 °C 2 min] x 12-21 cycles;
68 °C 5 min; 
4 °C f∞

 

 


SECTION: Final Bead Purification
29. Add 30 µL of thoroughly resuspended   to the reaction mix. Mix well by pipetting. Incubate for 5 min at room temperature.

SECTION: Final Bead Purification
30.   Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 5 min or until the supernatant is completely clear.

SECTION: Final Bead Purification
31. 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.
SECTION: Final Bead Purification
32. Add 15 µL of thoroughly resuspended SPRI beads to the retained supernatant from step #31. Mix well by pipetting. Incubate for 5 min at Room temperature.

SECTION: Final Bead Purification
33. Pellet beads by placing the sample tubes on a magnetic rack. Allow the beads to collect for 5 min or until the supernatant is completely clear.

SECTION: Final Bead Purification
34. Leaving the sample tubes on the magnetic rack, discard clear supernatant. Take care to not disturb the pelleted beads.
SECTION: Final Bead Purification
35. 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 30 s incubation remove and discard the supernatant.

SECTION: Final Bead Purification
36. 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. 
SECTION: Final Bead Purification
37. 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 2 min at Room temperature.

SECTION: Final Bead Purification
38. Place the sample tubes back on the magnetic plate and allow beads to pellet. Transfer 17 µL of the supernatant to a fresh tube. 

SECTION: Final Bead Purification
39. Samples are now ready for quality control, quantification, pooling, and sequencing.

Safe stopping point: samples can be stored at -20 °C


 

SECTION: First Bead Purification
13. 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 2 min at Room temperature

SECTION: [Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
40. Quantify samples using the Qubit dsDNA High Sensitivity Kit or with a BioAnalyzer High Sensitivity DNA kit.
SECTION: [Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
41. 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.
SECTION: [Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
42. 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. 
SECTION: [Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
43. 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). 
SECTION: [Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
44. 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).
SECTION: [Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
45. Samples are now ready for quality control, quantification, pooling, and sequencing.

Safe stopping point: samples can be stored at -20 °C