TITLE: X-ClickSeq: Custom-Primed Protocol with Single Indexing using ClickSeq Kit
AUTHORS: Andrew Routh, Elizabeth Jaworski

[DESCRIPTION]
ClickSeq is a simple, fragmentation-free method for the synthesis of Next-Generation Sequencing (NGS) libraries. ClickSeq derives its name by using ‘Click-Chemistry‘ in the place of common ligation enzymes to ‘click-ligate’ nucleic acids to sequencing adaptors – an essential and often problematic step in the synthesis of Next-Generation Sequencing cDNA libraries. 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. The modified nucleotides are stochastically incorporated into the nascent cDNA, 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. 

ClickSeq is a highly flexible and modular platform for NGS library synthesis where both RNA and DNA templates can be used as input material. ClickSeq offers many advantages over standard RNA NGS protocols as click-chemistry is utilized to attach on 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. Additionally, since there are no fragmentation steps typically required in common RNA-seq approaches and because in vitro template switching during RT is limited by the chain terminating azido-nucleotides, ClickSeq offers the benefit of ultra-low artifactual chimera rate, with only 3 chimeric events per million reads.

X-ClickSeq kits provide the same reagents as the ClickSeq kits, except that no primers are provided for the Reverse Transcription step. Rather, the user may use their own RT primer as per the requirements of their own assay. For example, a 9N random primer might be used instead of a 6N random primer; or a set of SARS-CoV-2 targeting tiled-primers might be used (see Jaworski et al. eLife, 2021). All other aspects of the library prep are otherwise identical to ClickSeq. The data analysis pipeline will also be altered as per the assay design. 

[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: RNA is reverse transcribed by user-provided primer(s) with a partial p7 Illumina adaptor sequence. The presence of AzNTPs stochastically terminates cDNA chain extension 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 all components of the PCR amplification reaction from the completed barcoded 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 sense laboratory precautions to minimize introducing contamination to your samples and follow procedures as written to ensure good yields.

RT-Primer Design
The X-ClickSeq kits purposefully omit any primer in the x-Primer Mix (xPM) used in the reverse transcription step. This allows users to provide their own bespoke primer(s), as per the needs of their specific project. To be compatible with the downstream reagents and oligos, these RT primers must contain first a partial i7 sequence, followed by the template-annealing region. For example, the random/6N primer included in the standard ClickSeq kit is:
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN

Thus, the user can substitute the ‘NNNNNN’ region with any sequence of interest, and/or provide many template-specific/tiled primers. The user-provided primer(s) are added to the reaction at step 3.
As a result, the X-ClickSeq kit provides users with the flexibility to design their own ClickSeq-based assays. 

The user-provided primer(s) must be resuspended in either nuclease-free water or Tris-buffer, but must not contain EDTA or other chelators. 

The user-provided primer(s) should be between 10-100µM, or can be optimized on a per-sample or application-specific basis. Template-specific or Tiled primers should generally not exceed 10µM, but must be validated on a case-by-case basis. 


RNA Handling
Work in an RNase-free environment; use RNase inhibitors to decontaminate your workspace. Follow standard aseptic techniques.
Wear PPE (gloves, lab coat, 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). We recommend you do NOT use carrier RNA during the extraction process. These will negatively impact your final sequencing data as the carrier RNA will also be sequenced.
When using ClickSeq for RNAseq applications, we recommend using protocols that do not co-purify genomic DNA, since this may also provide a substrate for the RT reaction. If available, complete the specified DNase I treatment during RNA extraction. 
The random primer of this ClickSeq kit will generate cDNA fragments off any nucleic acid in your sample. WHAT YOU PUT IN THE INTIAL REACTION (Step #1) IS WHAT YOU WILL SEQUENCE. Make sure to purify your samples prior to starting this protocol (i.e. rRNA deplete, polyA-select, or another purification method that suits your needs).
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 10ng up to 4µg of 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 >100ng of RNA. Below this value, additional PCR cycles are required that will result in PCR duplication and increased adaptor-dimers in the final library.
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 #5 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 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-1 µg of input RNA to 10 µL using nuclease free water.

 

SECTION: Reverse Transcription and RNA Removal
3. Add 1 µL of user-provided primer(s) to the diluted RNA. Mix well.

 

SECTION: Reverse Transcription and RNA Removal
4. 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
6. Add 7 µL of the RT master mix to each reaction and pipette to mix.

SECTION: Reverse Transcription and RNA Removal
7. 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
8. [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
9. 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
10. 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
11. Leaving the sample tubes on the magnetic rack, discard clear supernatant taking care to not disturb the pelleted beads.
SECTION: First Bead Purification
12. 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
13. Repeat the EtOH washing step (#12) 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
15. 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
16. Add 15 µL of Click Mix (CM) to each sample. Pipette to mix, taking care to not introduce air bubbles.

 

SECTION: Click-Ligation
17. 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
18. 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
5. 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
19. 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
20. 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
21. Leaving the sample tubes on the magnetic rack, discard clear supernatant. Take care to not disturb the pelleted beads.
SECTION: Second Bead Purification
22. 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
23. Repeat the EtOH wash step (#22) 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
24. 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
25. 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
26. 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
27. Add 25 µL of   to each sample tube.

SECTION: PCR Library Amplification
28. Using a unique Index Primer per sample, add 15 µL of each respective i7 index primer (Index Primer D701-D712) to each sample tube. Pipette to mix. Take note of which index was used for each sample. 

 
 

SECTION: PCR Library Amplification
29. 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
30. 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
31.   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
32. 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
33. Add 15 µL of thoroughly resuspended SPRI beads to the retained supernatant from step #32. Mix well by pipetting. Incubate for 5 min at Room temperature.

SECTION: Final Bead Purification
34. 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
35. Leaving the sample tubes on the magnetic rack, discard clear supernatant. Take care to not disturb the pelleted beads.
SECTION: Final Bead Purification
36. 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
37. Repeat the EtOH wash step (#36) 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
38. 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
39. 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
40. 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
14. 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)
41. 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)
42. 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)
43. 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)
44. Using a clean gel knife and referencing the DNA ladder, excise the gel between 300 and 600bp (or as required per the user’s assay). 
SECTION: [Optional] Agarose Gel DNA Extraction Protocol (if tighter fragment size distribution is required)
45. 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)
46. Samples are now ready for quality control, quantification, pooling, and sequencing.

Safe stopping point: samples can be stored at -20 °C 
SECTION: Reverse Transcription and RNA Removal
2. Add 2 µL of x-Primer Mix (xPM) to the diluted RNA. Mix well.