Single-cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategy

Gyeong-Jin Shin; Byung-Han Choi; Hye Hyeon Eum; Areum Jo; Nayoung Kim; Huiram Kang; Dongwan Hong; Jiyoung Joan Jang; Hwi-Ho Lee; Yeon-Su Lee; Yong Sun Lee; Hae-Ock Lee

doi:10.1371/journal.pone.0301562

Peer Review History

Original SubmissionMarch 18, 2024
6 May 2024 Decision Letter - Abozar Ghorbani, Editor PONE-D-24-10785Single cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategyPLOS ONE Dear Dr. Lee, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 20 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Reviewer #1: No Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: I have questions regarding the experimental design that require clarification from the authors? 1.Limited ncRNA Coverage: While the developed methodology represents a significant step in addressing the challenge of profiling non-coding RNAs (ncRNAs) transcribed by RNA polymerase III (Pol III), it may still have limitations in capturing all types of ncRNAs. It's possible that other classes of ncRNAs or specific transcripts may not be effectively captured using this approach. How do the authors plan to address these potential limitations? 2. Oligo Tag Specificity: The authors note that the identification of oligo tags showed limited target specificity, implying that the oligo-tagged primer approach may not be completely specific to the target ncRNA. This raises concerns about potential off-target effects or noise in the sequencing data. Have the authors encountered off-target effects during their experimentation? How did they evaluate the specificity of the tags for other ncRNAs? 3. Validation and Reproducibility: While the methodology was applied to three different cell lines with varying levels of nc886 expression, further validation across a larger and more diverse set of cell types, tissues, or biological conditions would enhance the robustness and generalizability of the findings. Additionally, ensuring reproducibility of the results across different experimental setups and laboratories is essential. The authors' validation of this approach and the reproducibility of the results are important aspects to consider. How authors will address it? Reviewer #2: The manuscript entitled “Single cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategy” have provided a measurement protocol for nc886 and then applied this protocol in three cell lines which express high, low, and zero levels of nc886. While it is a very valuable and interesting work, the manuscript raises several serious concerns to support its conclusion: 1. The authors must enhance the manuscript language by getting it reviewed by an English language expert to make it convenient for the readers to comprehend. The manuscript needs many corrections in terms of grammar and spelling. 2. In the method, the author should specify the cell type used for each cell line and the primers utilized in the Real-time PCR. 3. The discussion is inadequately written. It is essential to compare the results with previous studies. 4. What are the planned future experiments? 5. The complete results section description needs to be detailed. 6. The protocol's validation should involve a larger number of cell lines. 7. At the end of the manuscript, the conclusion is not provided. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: Dear Editor.docx https://doi.org/10.1371/journal.pone.0301562.r001
Revision 1
19 Jun 2024 Author Response June 14, 2024 Abozar Ghorbani, PhD Academic Editor, PLOS ONE Dear Dr. Abozar, We would like to thank you and the reviewers for the valuable feedback on our manuscript titled “Single cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategy”. We have carefully considered the comments and suggestions and revised the manuscript accordingly. Point-by-point responses Reviewer #1: I have questions regarding the experimental design that require clarification from the authors? 1.Limited ncRNA Coverage: While the developed methodology represents a significant step in addressing the challenge of profiling non-coding RNAs (ncRNAs) transcribed by RNA polymerase III (Pol III), it may still have limitations in capturing all types of ncRNAs. It's possible that other classes of ncRNAs or specific transcripts may not be effectively captured using this approach. How do the authors plan to address these potential limitations? : We agree with the reviewer that there are limitations and challenges in capturing other RNAs. Our method is not suitable for RNAs that cannot be amplified with gene specific primers. We chose the strategy of barcoded gene-specific primers (GSPs) because of its simplicity and expandability. This strategy can be applied to other ncRNAs by simply using different GSPs. Therefore, we expect that our experience with nc886 will provide valuable insights and tips for working with other ncRNAs. In our scRNA-seq of nc886, we encountered non-specific priming, but were able to map the sequencing reads successfully. Our approach to overcoming this issue can be applied if a similar problem arises in scRNA-seq of other ncRNAs. Additionally, our study clearly highlighted the need to improve the scRNA-seq protocol. In our revised manuscript, we have extensively discussed potential directions and solutions for improvement (pages 14-15, lines 287-306 in the revised version), all of which can be applied to other classes of ncRNAs. 2. Oligo Tag Specificity: The authors note that the identification of oligo tags showed limited target specificity, implying that the oligo-tagged primer approach may not be completely specific to the target ncRNA. This raises concerns about potential off-target effects or noise in the sequencing data. Have the authors encountered off-target effects during their experimentation? How did they evaluate the specificity of the tags for other ncRNAs? : The off-target effects were demonstrated by the sequencing data (Figure 3A, right side: upper bar graph), showing that only a small fraction of the feature barcode counts corresponded to the nc886 gene. Our in vitro experiment (newly added Figure S1) also indicated while primers bound to nc886, non-specific priming was amplified during the cDNA amplification step. We addressed this by extracting sequences located after the feature barcode and counting those mapped to nc886. As detailed in lines 287-295 on pages 14-15 of the revised version, primer design is for nc886 is particularly limited and challenging. For other ncRNAs, especially those of sufficient length, the best strategy is to design multiple primers, select the best one through preliminary in vitro experiments (such as Figure S1), and then proceed with scRNA-seq. In the worst case, as our experience with nc886 suggests, sequences can be extracted. 3. Validation and Reproducibility: While the methodology was applied to three different cell lines with varying levels of nc886 expression, further validation across a larger and more diverse set of cell types, tissues, or biological conditions would enhance the robustness and generalizability of the findings. Additionally, ensuring reproducibility of the results across different experimental setups and laboratories is essential. The authors' validation of this approach and the reproducibility of the results are important aspects to consider. How authors will address it? : As the reviewer pointed out, we also recognize that it is important to apply this technique to many cell types, tissues, and biological conditions to enhance the robustness and generalizability of our findings. Collecting and comparing more data will not only provide biological information, but also help validate the modified scRNA protocol. However, we are more focused on optimizing the protocol before performing scRNA-seq in many samples. Once optimized, we plan to perform scRNA-seq experiments in many biological samples. ================================================================== Reviewer #2: The manuscript entitled “Single cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategy” have provided a measurement protocol for nc886 and then applied this protocol in three cell lines which express high, low, and zero levels of nc886. While it is a very valuable and interesting work, the manuscript raises several serious concerns to support its conclusion: 1. The authors must enhance the manuscript language by getting it reviewed by an English language expert to make it convenient for the readers to comprehend. The manuscript needs many corrections in terms of grammar and spelling. : In accordance with the reviewers' comments, our manuscript has been edited by a professional English editing service. The certificate is attached. 2. In the method, the author should specify the cell type used for each cell line and the primers utilized in the Real-time PCR. : We specified the cell type used for each cell line and the primers utilized in the Real-Time (RT) PCR (in the revised Fig. 1A). 3. The discussion is inadequately written. It is essential to compare the results with previous studies. : We have reorganized and rewritten the discussion into three sections. In the first section, we have summarized our findings, highlighting both the benefits and the limitations. In the second section, we have discussed the limitations in detail and proposed potential strategies to address them. The final section compares our study with previous single-cell studies on ncRNAs and discusses further research directions and potential extensions. 4. What are the planned future experiments? : Based on the points discussed in the Discussion section, we are planning experiments to address the current limitations identified. One significant challenge is the inefficient amplification of nc886-GSP products. We have proposed several strategies for improvement to mitigate issues of non-specificity (lines 296-306, page 15). These strategies are designed to enhance the efficiency of capturing ncRNA. After optimizing the protocol, we intend to apply our method to a broad spectrum of cell types, tissues, and biological conditions. Specifically, we are interested in exploring the role of nc886 in colon cancer and investigating how its heterogeneous expression correlates with the gene expression phenotypes of tumor cells. 5. The complete results section description needs to be detailed. : In response to the reviewer’s critique, we revised the Results section to provide clearer descriptions of the figures. Specific changes include: Page 10, lines 180-181 The cell lines were specifically mentioned (WPMY-1, Hep3B-nc886, HEK293T), and detailed descriptions of the cell lines were added. Page 10, lines 182-184 Methods for verifying nc886 expression levels using qRT-PCR and Northern hybridization were added. Page 10, lines 185-195 We provided comprehensive information about nc886-GSP and detailed the in vitro validation performed before implementing the modified scRNA-seq. Page 10, lines 194-196 We included information confirming the correct synthesis of the nc886 cDNA, as verified by PCR results and sequencing, which are shown in the revised supplementary Fig. S1. These revisions offer a clearer and more specific explanation to assess the validity of nc886 detection and support the feasibility of using GSPs. Page 12, lines 231-247 We updated the Fig 3A and improved the alignment between the figure and the text descriptions in detail. Page 13, lines 249-250 (title), 251-271 (contents) We modified the section title and emphasized the integrated analysis of nc886 and 5’ gene expression data. page 13, lines 268-271 We proposed that the heterogeneity of nc886 expression levels might also be relevant to other samples, such as colon cancer, offering additional biological insights. For additional changes, please refer to the tracked version of the revised manuscript. 6. The protocol's validation should involve a larger number of cell lines. We agree with the reviewer that this protocol requires further validation with a larger number of cell lines. Expanding the range of cell lines will enable us to quantify the gene expression levels of nc886 and match them with single-cell analysis. We added a discussion section on this point (page 16, lines 320-322 in the revised version). 7. At the end of the manuscript, the conclusion is not provided. In the revision, we added a Conclusion section after the Discussion. (page 16, lines 328-333 in the revised version). Attachments Attachment Submitted filename: [Finalize] Response to the reviewers_240618.docx https://doi.org/10.1371/journal.pone.0301562.r002
8 Jul 2024 Decision Letter - Abozar Ghorbani, Editor Single cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategy PONE-D-24-10785R1 Dear Dr. Lee, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The author's responses were highly compelling, and this data is sufficient for publication in support of the newly developed protocol. This manuscript is well-written and does not require further revision. Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0301562.r003
Formally Accepted
16 Aug 2024 Acceptance Letter - Abozar Ghorbani, Editor PONE-D-24-10785R1 PLOS ONE Dear Dr. Lee, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Abozar Ghorbani Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0301562.r004

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