Isolating and cryopreserving pig skin cells for single-cell RNA sequencing study

Li Han; Carlos P. Jara; Ou Wang; Yu Shi; Xinran Wu; Sandra Thibivilliers; Rafał K. Wóycicki; Mark A. Carlson; William H. Velander; Eliana P. Araújo; Marc Libault; Chi Zhang; Yuguo Lei

doi:10.1371/journal.pone.0263869

Peer Review History

Original SubmissionMarch 22, 2021
13 May 2021 Decision Letter - Nazmul Haque, Editor PONE-D-21-09015 Isolating and Cryo-Preserving Pig Skin Cells for Single Cell RNA Sequencing Study PLOS ONE Dear Dr. Lei, 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 June 04, 2021. 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Nazmul Haque Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: 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: In this study, Han et al describe a method for isolating and preserving primary porcine skin cells for subsequent analysis by single cell RNA sequencing. They demonstrate that the cryopreservation process o purified cells minimally impacts cell viability and the transcriptional profile of the isolated cells. Comments: 1) There are some typos in Figure 1B that should be corrected (Cry-thaw, FACs, MACs) 2) Figure 4 and Figure 5 are very duplicative. The figures could be combined, or figure 4 moved to supplemental data (or deleted). 3) Figure 3a: the axis of the plot need to be labeled more clearly. Assuming that the data shown is the relative gene expression. Log transforming the data on such a plot would be a more conventional way to display the data, and highlighting some important genes would help with orientation. 4) The description of the trajectory analysis could use some refinement. As currently written the data is presented as demonstrating that cell differentiation occurs in both fresh and frozen cells. A more correct/helpful description of the data would be something along the lines of "the data quality obtained from both fresh and frozen cells was of sufficient quality to allow transcriptional trajectory/pseudotime analysis" 5) Population identification/annotation is a very important step in scRNAseq analysis, especially for novel tissue types and "non-standard" model systems. It might be helpful to move some of the linage-defining gene expression data from supplemental data to a primary figure, and perhaps use a dot plot to highlight key genes from each population. Reviewer #2: The current manuscript entitled ‘Isolating and Cryo-Preserving Pig Skin Cells for Single Cell RNA Sequencing Study’ is well written. However, there are some points need to be addressed before publication – 1# If I want to summarize the content of this manuscript, there would be three key points – i) Isolation of single cell from Pig skin, ii) Cryo-preservation of that single cell and iii) RNA sequencing of that single pig skin cell. scRNA-seq is a common and established method of gene expression analysis if the single cell is available. Thus, the main part of this manuscript is the isolation and preservation of single pig skin cells. Unfortunately, the authors highlighted scRNA-seq in the abstract not the isolation methodology. In the Introduction section the authors did the same approach. They need to rewrite the Abstract and Introduction in a more presentable format. Additionally, the last paragraph of Introduction needs to be removed. Introduction should not contain any methodology or results. Instate of that the authors need to present their aim of this research. 2# Which one is correct – pigskin or pig skin? Authors need to follow only one style throughout the manuscript. 3# In the Materials and Methods, the authors claimed only Methods. Does it compatible with Plos One? The first sub-heading in this section is entitled as ‘Pig skins’. Does it mean anything? It could be ‘Harvesting of pig skin’. Writing of a manuscript and writing of lab note is different. Authors are suggested to recheck all the sub-headings in this manuscript and change them in the more presentable format, if required. Additionally, they are suggested to follow the international format of writing the company names for any chemicals and instruments throughout the manuscript. 4# The isolation of single cell from pig skin is the main part of this manuscript. So, authors need to describe in detail procedure in the Materials and Methods. The present description is not satisfactory. 5# Author wrote ‘Libraries were sequenced using Illumina technology.’ Does they have sequencer in their Lab? If so, then provide the company name. If not, then mention the commercial service name. 6# Authors need to recheck the suitability of data deposition statement in the body text. If nor, they are requested to remove the ‘Data and code availability’ section from the text. 7# Which one is correct – liquid N2 or liquid N2? Authors need to follow only one style throughout the manuscript. Furthermore, they need to check for cryopreservation or cryo-preservation. And so on ………. 8# It is well established that 10% DMSO provides good preservation of live cells. The authors of this manuscript also used the same procedure. However, they want to claim that they did something new. They are requested to show every point they have modified in the general procedure. Additionally, several times they used the term ‘DMSO-based cryopreservation’. For example, they claim that DMSO-based cryopreservation preserved gene expression. What does it mean? Does it mean that among several cryopreservation techniques DMSO-based cryopreservation showed optimum result? Does it mean that DMSO-based cryopreservation is mandatory for pig skin cell? The authors need to show a comparative analysis among different preservation techniques if they want to claim so. Additionally, they need to provide a positive control (either human skin/rodent skin cells) to compare. 9# In summary, the authors isolated single cells from pig skin, cryopreserved and compare it with fresh cell in genomic level. Thus, the merit goes to the impact of preservation in genomic level. So far, this manuscript is pioneer to isolating single cells from pig skin. In this regards it has novelty. But if they want to show the impact of preservation then the present format of manuscript is not suitable. They need to add positive control and comparison data for different preservation techniques. And rewrite the manuscript in particular manner. 10# Rigorous English editing is required. ****** 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:** Adam T Waickman 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.0263869.r001
Revision 1
16 Sep 2021 Author Response Reviewer #1: 1) There are some typos in Figure 1B that should be corrected (Cry-thaw, FACs, MACs) Response: Revised. 2) Figure 4 and Figure 5 are very duplicative. The figures could be combined, or figure 4 moved to supplemental data (or deleted). Response: We moved Fig 4 to supplemental data (S2 Fig). 3) Figure 3a: the axis of the plot need to be labeled more clearly. Assuming that the data shown is the relative gene expression. Log transforming the data on such a plot would be a more conventional way to display the data, and highlighting some important genes would help with orientation. Response: Revised following the suggestion. 4) The description of the trajectory analysis could use some refinement. As currently written the data is presented as demonstrating that cell differentiation occurs in both fresh and frozen cells. A more correct/helpful description of the data would be something along the lines of "the data quality obtained from both fresh and frozen cells was of sufficient quality to allow transcriptional trajectory/pseudotime analysis" Response: Revised as suggested. 5) Population identification/annotation is a very important step in scRNAseq analysis, especially for novel tissue types and "non-standard" model systems. It might be helpful to move some of the linage-defining gene expression data from supplemental data to a primary figure, and perhaps use a dot plot to highlight key genes from each population. Response: Following the suggestion, we added Fig 5. Reviewer #2: 1# If I want to summarize the content of this manuscript, there would be three key points – i) Isolation of single cell from Pig skin, ii) Cryo-preservation of that single cell and iii) RNA sequencing of that single pig skin cell. scRNA-seq is a common and established method of gene expression analysis if the single cell is available. Thus, the main part of this manuscript is the isolation and preservation of single pig skin cells. Unfortunately, the authors highlighted scRNA-seq in the abstract not the isolation methodology. In the Introduction section the authors did the same approach. They need to rewrite the Abstract and Introduction in a more presentable format. Additionally, the last paragraph of introduction needs to be removed. Introduction should not contain any methodology or results. Instate of that the authors need to present their aim of this research. Response: Thanks for the comments. We revised the manuscript as suggested. 2# Which one is correct – pigskin or pig skin? Authors need to follow only one style throughout the manuscript. Response: Pig skin is used in the revised manuscript. 3# In the Materials and Methods, the authors claimed only Methods. Does it compatible with Plos One? The first sub-heading in this section is entitled as ‘Pig skins’. Does it mean anything? It could be ‘Harvesting of pig skin’. Writing of a manuscript and writing of lab note is different. Authors are suggested to recheck all the sub-headings in this manuscript and change them in the more presentable format, if required. Additionally, they are suggested to follow the international format of writing the company names for any chemicals and instruments throughout the manuscript. Response: Revised as suggested. 4# The isolation of single cell from pig skin is the main part of this manuscript. So, authors need to describe in detail procedure in the Materials and Methods. The present description is not satisfactory. Response: We added details of the isolation process. 5# Author wrote ‘Libraries were sequenced using Illumina technology.’ Does they have sequencer in their Lab? If so, then provide the company name. If not, then mention the commercial service name. Response: Revised as suggested. 6# Authors need to recheck the suitability of data deposition statement in the body text. If nor, they are requested to remove the ‘Data and code availability’ section from the text. Response: Removed from the text. 7# Which one is correct – liquid N2 or liquid N2? Authors need to follow only one style throughout the manuscript. Furthermore, they need to check for cryopreservation or cryo-preservation. And so on ………. Response: Changed all the “liquid N2” to “liquid N2”. In addition, we replaced “frozen” by “cryopreserved”. 8# It is well established that 10% DMSO provides good preservation of live cells. The authors of this manuscript also used the same procedure. However, they want to claim that they did something new. They are requested to show every point they have modified in the general procedure. Additionally, several times they used the term ‘DMSO-based cryopreservation’. For example, they claim that DMSO-based cryopreservation preserved gene expression. What does it mean? Does it mean that among several cryopreservation techniques DMSO-based cryopreservation showed optimum result? Does it mean that DMSO-based cryopreservation is mandatory for pig skin cell? The authors need to show a comparative analysis among different preservation techniques if they want to claim so. Additionally, they need to provide a positive control (either human skin/rodent skin cells) to compare. Response: Thanks for the comments. First, we changed the “DMSO-based cryopreservation” to “cryopreservation in 90% FBS +10% DMSO” to avoid the confusion. Second, we did not claim: 1) we invented a new cryopreservation method, or improved/modified the DMSO-based cryopreservation method; 2) the DMSO-based cryopreservation is better than other cell preservation methods. The goal of this work is to find, demonstrate, validate a robust method/protocol for isolating and preserving single pig skin cells for scRNA-Seq study. To our best knowledge, there have been no reports on how to prepare and preserve single cells from pig skin tissue for scRNA-Seq in the literature. We showed that using our reported method/protocol, high-quality scRNA-Seq data could be generated to identify the major skin cell types. We believe this report is very valuable for the skin research scientific community. 9# In summary, the authors isolated single cells from pig skin, cryopreserved and compare it with fresh cell in genomic level. Thus, the merit goes to the impact of preservation in genomic level. So far, this manuscript is pioneer to isolating single cells from pig skin. In this regards it has novelty. But if they want to show the impact of preservation then the present format of manuscript is not suitable. They need to add positive control and comparison data for different preservation techniques. And rewrite the manuscript in particular manner. Response: same as 8#. 10# Rigorous English editing is required. Response: Revised as the suggestion. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0263869.r002
13 Dec 2021 Decision Letter - Nazmul Haque, Editor PONE-D-21-09015R1Isolating and cryopreserving pig skin cells for single-cell RNA sequencing studyPLOS ONE Dear Dr. Lei, 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. Specifically, If the methods have not been developed by you then please refer to the article(s) from where you have adopted or modified these methods. Please submit your revised manuscript by Jan 27 2022 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Nazmul Haque Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] 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: (No Response) ******** 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: (No Response) Reviewer #2: A) In response to my query #8 the authors answered as follows – 1) We invented a new cryopreservation method, or improved/modified the DMSO-based cryopreservation method; Observation: The authors used three keywords invention, improvement and modification. Which one is appropriate? Do they really invent a new protocol??? I think they just modified the existing protocol for pig skin cell isolation. If so, then they need to let us know in which points they have changed. Furthermore, does the modification bring better result? If so, then they need to show us both results in a comparison manner. We need to understand that their modification is meaningful. 2) The DMSO-based cryopreservation is better than other cell preservation methods. Observation: We all agree with this statement. Everybody knows that the DMSO-based cryopreservation method is the best one. Then, what is new here the author is claiming. Why the authors are highlighting DMSO-based cryopreservation for pig cells? This would be a normal procedure for cell preservation. B) The reviewer is not satisfied with the answer for #9. The authors need to organize their manuscript in a manner that they just isolated single cells from pig skin. ****** 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:** Adam Waickman 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.0263869.r003
Revision 2
19 Jan 2022 Author Response Reviewers' comments: Reviewer #2: A) In response to my query #8 the authors answered as follows – 1) We invented a new cryopreservation method, or improved/modified the DMSO-based cryopreservation method; Observation: The authors used three keywords invention, improvement and modification. Which one is appropriate? Do they really invent a new protocol??? I think they just modified the existing protocol for pig skin cell isolation. If so, then they need to let us know in which points they have changed. Furthermore, does the modification bring better result? If so, then they need to show us both results in a comparison manner. We need to understand that their modification is meaningful. 2) The DMSO-based cryopreservation is better than other cell preservation methods. Observation: We all agree with this statement. Everybody knows that the DMSO-based cryopreservation method is the best one. Then, what is new here the author is claiming. Why the authors are highlighting DMSO-based cryopreservation for pig cells? This would be a normal procedure for cell preservation. Response: There is some miscommunication here. We strongly agree with the reviewer about the observations. In our response to to query #8, we said: “Second, we did not claim: 1) we invented a new cryopreservation method, or improved/modified the DMSO-based cryopreservation method; 2) the DMSO-based cryopreservation is better than other cell preservation methods” We said “we did not claim”. B) The reviewer is not satisfied with the answer for #9. The authors need to organize their manuscript in a manner that they just isolated single cells from pig skin. Response: Sorry that we did not have a clear answer in our last response. We did re-ogranize the manuscript in a manner suggested by the reviewer in the previous revision. Again, thanks for all the suggestions! Attachments Attachment Submitted filename: Response to Reviewers(2).docx https://doi.org/10.1371/journal.pone.0263869.r004
31 Jan 2022 Decision Letter - Nazmul Haque, Editor Isolating and cryopreserving pig skin cells for single-cell RNA sequencing study PONE-D-21-09015R2 Dear Dr. Lei, 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 #2: (No Response) ******** 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 #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: (No Response) ****** 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 #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 #2: No https://doi.org/10.1371/journal.pone.0263869.r005
Formally Accepted
8 Feb 2022 Acceptance Letter - Nazmul Haque, Editor PONE-D-21-09015R2 Isolating and cryopreserving pig skin cells for single-cell RNA sequencing study Dear Dr. Lei: 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.0263869.r006

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