A few questions about experimental protocol

[jingkun12137]

Hello,
Thank you for developing this excellent tRNA sequencing protocol. I'm a PhD student following your Direct tRNA-seq library preparation protocol and have a question about reagent substitutions.
Due to availability constraints in our laboratory, I'm using the following reagents instead of those specified in the original protocol:
Substitutions I'm using:

50% PEG 8000 (prepared in DEPC-treated water, 8 μL) instead of NEB PEG 8000 (Cat. No. B1004S, 4 μL)
Agencourt AMPure XP (Beckman Coulter, Cat. No. A63880)
tRNA purification beads (BioDynami, Cat. No. 40054S)
T4 RNA ligase 2 and buffer (NEB, Cat. No. M0239L)

Additional modification:

Performing ligation reactions at 25°C (temperature-controlled) instead of room temperature due to elevated ambient temperature in our lab

Could you please confirm whether these reagent substitutions are compatible with your protocol? I'm particularly concerned about the PEG 8000 substitution and the magnetic bead replacement.
Any guidance would be greatly appreciated.
Thank you for your time and assistance.

[jayhesselberth]

Are you using the AMPure beads instead of the tRNA purification beads?

Other than that I don't think your changes would impact ligation yield.

[jingkun12137]

Thank you very much for your reply. According to what you mentioned in the protocol, I used tRNA purification beads (BioDynami, Cat. No. 40054S) after connecting splint adapter and RTA. After connecting RLA, used Agencourt AMPure XP (Beckman Coulter, Cat. No. A63880). The product can be observed by electrophoresis after connecting splint adapter and RTA, but the product cannot be observed after connecting RLA.

[lkwhite]

We never run RLA-ligated material on a gel. If the sizes look correct after RTA addition I move forward with sequencing, because you have much less material after the final ligation and cleanup, and the helicase-loaded adapter is always the limiting reagent in ONT’s kits.

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On Wed, Jul 30, 2025 at 2:07 AM jingkun12137 ***@***.***> wrote: *jingkun12137* left a comment (rnabioco/tRNA004#13) <#13 (comment)> Thank you very much for your reply. According to what you mentioned in the protocol, I used tRNA purification beads (BioDynami, Cat. No. 40054S) after connecting splint adapter and RTA. After connecting RLA, used Agencourt AMPure XP (Beckman Coulter, Cat. No. A63880). The product can be observed by electrophoresis after connecting splint adapter and RTA, but the product cannot be observed after connecting RLA. — Reply to this email directly, view it on GitHub <#13 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AB63UITTHRW7L4LZZXBMVU33LB4K7AVCNFSM6AAAAACCPWW5RCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZTCMZVGI3DQNZXGM> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[jingkun12137]

Due to laboratory equipment limitations, I can only perform RNA gel electrophoresis using TBE-Urea precast gels. The attached images show my electrophoresis results. Similarly, due to equipment constraints, I can only store the samples at 4°C for approximately 24 hours after RTA ligation before shipping them to the sequencing company for ONT sequencing.
When the company received my RTA ligation products, they informed me that their measurements showed the product concentration was nearly zero. I suspect the issue occurred either during the RTA ligation step or during the shipping process, though I cannot definitively confirm whether the SA and RLA ligation was successful.

I would like to express my sincere gratitude once again for the protocol you shared. If you could provide any insights or suggestions regarding this matter, I would be immensely grateful.

[lkwhite]

Your splint adapter ligation looks like it's working but this gel doesn't look like it validates anything after that. Just to confirm, you are doing this in the order:

1. ligate splint adapters (SA)
2. ligate first ONT adapters (RTA)
3. ligate second ONT adapters (RLA)

Asking since your final lanes are ordered 1, 3, 2 in my list above.

[jingkun12137]

I sincerely apologize for the error I made in the image. I did indeed follow your protocol description exactly as stated - connecting RTA first, then RLA. The corrected image is shown as attached. Perhaps I had already failed during the first step of connecting the splint adapters.

When connecting the splint adapters, I used 600 ng of small RNA for ligation (dissolved in 6 μL nuclease-free water) + 2 μL T4 RNA Ligase 2 Buffer (NEB) + 8 μL 50% PEG8000 (solvent: ddH2O) + 2 μL T4 RNA Ligase 2 (NEB) + 1 μL Murine RNase Inhibitor (NEB, M0314S) + 1 μL splint adapters. After thorough mixing, the reaction was incubated at 25°C for 30 minutes.

Does this ligation reaction appear to be correct? If so, there may be an issue with the reagents I purchased, and I will need to repurchase them and repeat the experiment.

[lkwhite]

Got it. I think you need to be able to convince yourself you see a shift on RTA ligation before you move forward to RLA. Take a look at figure S1D in our preprint for an example of what this shift looks like on a 10% denaturing gel. Again I'm not sure whether you'll be able to see much after RLA ligation, but I would focus your immediate troubleshooting on the previous step.

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On Mon, Aug 4, 2025 at 8:19 PM jingkun12137 ***@***.***> wrote: *jingkun12137* left a comment (rnabioco/tRNA004#13) <#13 (comment)> RNA.electrophoresis.jpg (view on web) <https://github.com/user-attachments/assets/78b1a228-e17f-4660-b15b-6720819d3960> I sincerely apologize for the error I made in the image. I did indeed follow your protocol description exactly as stated - connecting RTA first, then RLA. The corrected image is shown as attached. Perhaps I had already failed during the first step of connecting the splint adapters. When connecting the splint adapters, I used 600 ng of small RNA for ligation (dissolved in 6 μL nuclease-free water) + 2 μL T4 RNA Ligase 2 Buffer (NEB) + 8 μL 50% PEG8000 (solvent: ddH2O) + 2 μL T4 RNA Ligase 2 (NEB) + 1 μL Murine RNase Inhibitor (NEB, M0314S) + 1 μL splint adapters. After thorough mixing, the reaction was incubated at 25°C for 30 minutes. Does this ligation reaction appear to be correct? If so, there may be an issue with the reagents I purchased, and I will need to repurchase them and repeat the experiment. — Reply to this email directly, view it on GitHub <#13 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AB63UIVJKYBI5MFQ42EPCTD3MAIDTAVCNFSM6AAAAACCPWW5RCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZTCNJTGA2DKNRWGQ> . You are receiving this because you commented.Message ID: ***@***.***>

[jingkun12137]

Thank you very much for your suggestions. In my upcoming experiments, I will focus on troubleshooting the RTA ligation step.