Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag

Yasuhiro Fujiwara; Yuji Tanno; Hiroki Sugishita; Yusuke Kishi; Yoshinori Makino; Yuki Okada

doi:10.1371/journal.pone.0259846

Peer Review History

Original SubmissionMay 13, 2021
16 Jul 2021 Decision Letter - Nazmul Haque, Editor PONE-D-21-13514 Preparation of "stress-free" concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag PLOS ONE Dear Dr. Fujiwara, 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 Aug 30 2021 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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Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 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: Yes ****** 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: The authors test different concanavalin A coated magnetic beads for CUT&RUN and CUT&Tag and find that the commercial beads recommended for these protocols are not ideal in that they may cause clumping and sticking to the sides of tubes. The authors test various versions of Dynabeads and find that the hydrophobic product solves the problem. Derivatizing Dynabeads with ConA is fast and simple, and given the improved results, it should be easily adopted by labs using these methods, and is likely to become popular. The work is clearly presented and conservatively interpreted. Reviewer #2: The manuscript “Preparation of "stress-free" concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag” comparing different formulations of magnetic beads for cell handling in CUT&Tag chromatin profiling. The basic CUT&Tag protocol for chromatin profiling is increasingly popular in many laboratories. In standard protocols commercial magnetic beads coupled to concanavalin A are used to capture cells for processing. The authors develop a procedure for making bead preparations from commercial strptavidin-coated magnetic beads and commercial biotinylated concanavalin A. The authors describe beads that are pretty suitable for CUT&Tag, with better suspension characteristics. However, they claim that these bead preparations improve the data quality of CUT&Tag chromatin profiling. This claim needs additional analysis for support. Line 44-45 states that “...the 45 MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3.” However, the data presented in Figure 4 shows that MyOne T1 beads have substantially increased background signal, seemingly twice as much as the BioMag, with only best a ~10% gain in signal. This is most obvious in Figure 4C and 4D. Quantitation of signal and background should be shown to support the conclusion that data quality is improved. The meaning of “stress-free” in the title is not clear and should be removed. At first this seemed to refer to a more gentle reagent for sensitive cells, but this term is only mentioned in the last sentence “...we propose that the home-made con A-conjugated Dynabeads magnetic beads (i. e. MyOne T1) can be a "stress-free" alternative choice for CUT&RUN and CUT&Tag methods.” Is this stress-free for the experimenter? If not, it should be clarified. ****** 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: Yes:** Steven Henikoff 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. https://doi.org/10.1371/journal.pone.0259846.r001
Revision 1
10 Oct 2021 Author Response Response to reviewers’ comments August 30, 2021 Dear Reviewers, We thank the reviewers for their careful reading of the manuscript and their thoughtful comments and suggestions. Also, we greatly appreciate the editor's decision to give us a chance to submit the revised manuscript. Our point-to-point responses to editorial and reviewers' comments are shown below. We believe our revised manuscript would suffice the reviewer's concerns. In the revised manuscript, the sentences added or changed are shown as marked-up of Track Change function of Word in the main text titled "Revised Manuscript with Track Changes". It would be greatly appreciated if the editorial office and reviewers could consider this revision. Reviewer #1: The authors test different concanavalin A coated magnetic beads for CUT&RUN and CUT&Tag and find that the commercial beads recommended for these protocols are not ideal in that they may cause clumping and sticking to the sides of tubes. The authors test various versions of Dynabeads and find that the hydrophobic product solves the problem. Derivatizing Dynabeads with ConA is fast and simple, and given the improved results, it should be easily adopted by labs using these methods, and is likely to become popular. The work is clearly presented and conservatively interpreted. Response: We appreciate the positive evaluation, which requires no specific response. Reviewer #2: The manuscript “Preparation of "stress-free" concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag” comparing different formulations of magnetic beads for cell handling in CUT&Tag chromatin profiling. The basic CUT&Tag protocol for chromatin profiling is increasingly popular in many laboratories. In standard protocols commercial magnetic beads coupled to concanavalin A are used to capture cells for processing. The authors develop a procedure for making bead preparations from commercial strptavidin-coated magnetic beads and commercial biotinylated concanavalin A. The authors describe beads that are pretty suitable for CUT&Tag, with better suspension characteristics. However, they claim that these bead preparations improve the data quality of CUT&Tag chromatin profiling. This claim needs additional analysis for support. Response: We appreciate the reviewer for the positive evaluation and valuable comments. As suggested, we re-analyzed the data and added some new data, which improved the manuscript. Comment #1: Line 44-45 states that “...the 45 MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3.” However, the data presented in Figure 4 shows that MyOne T1 beads have substantially increased background signal, seemingly twice as much as the BioMag, with only best a ~10% gain in signal. This is most obvious in Figure 4C and 4D. Quantitation of signal and background should be shown to support the conclusion that data quality is improved. Response: We appreciate for bringing the background issue to our attention. Some of the figure panels, especially old Fig4C, can mislead the readers. Using our con-A beads, more antibody was presumably introduced into cells efficiently, probably increasing the antibody's non-specific binding. Therefore, as you suggested, our data clearly showed higher background in T1 samples than in BioMag samples, especially at those peaks with high enrichment. However, those background signals were considerably low compared with H3K4me3 reads, and the S/N ratio is remarkably high. Because the y axis of the line plot in the old Fig4C was shown in the log scale, we changed it to linear (panel E in the new figure 4). We further re-analyzed the T1 and BioMag peaks and found that at T1-specific peaks, both T1 and BM samples showed read enrichment at a certain level. However, the S/N ratio for the BM sample was lower and not enough to be detected as a peak (new Fig4C, D). Further experiments are required to answer whether these peaks have biological meanings or background noises remain elusive. Comment #2: The meaning of “stress-free” in the title is not clear and should be removed. At first this seemed to refer to a more gentle reagent for sensitive cells, but this term is only mentioned in the last sentence “...we propose that the home-made con A-conjugated Dynabeads magnetic beads (i. e. MyOne T1) can be a "stress-free" alternative choice for CUT&RUN and CUT&Tag methods.” Is this stress-free for the experimenter? If not, it should be clarified. Response: We agree with the reviewer's suggestions and removed the "stress-free" from the title. We instead added 'optimized' to emphasize the advantages of the beads. In addition, the last paragraph of the discussion part was changed from “...we propose that the home-made con A-conjugated Dynabeads magnetic beads (i. e. MyOne T1) can be an alternative "stress-free" choice for CUT&RUN and CUT&Tag methods.” to “...we propose that the home-made con A-conjugated Dynabeads magnetic beads (i. e. MyOne T1) can be an alternative "stress-free" choice for CUT&RUN and CUT&Tag methods to avoid unwanted clumping and sticking to the sides of tubes. Furthermore, preparation of the beads can be done fast and simple, thus most likely improve the quality of CUT&Tag or CUT&RUN results.”. This summarizes why and how this method improves CUT&Tag method with minimal addition of steps. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0259846.r002
28 Oct 2021 Decision Letter - Nazmul Haque, Editor Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag PONE-D-21-13514R1 Dear Dr. Fujiwara, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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. Kind regards, Nazmul Haque Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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 authors' responses are satisfactory. This contribution represents a useful modification of a method that many people are beginning to use. The manuscript is ready for publication in my opinion. 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: Yes:** Steven Henikoff Reviewer #2: No https://doi.org/10.1371/journal.pone.0259846.r003
Formally Accepted
2 Nov 2021 Acceptance Letter - Nazmul Haque, Editor PONE-D-21-13514R1 Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag Dear Dr. Fujiwara: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Nazmul Haque Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259846.r004

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