miR‐AB, a miRNA‐based shRNA viral toolkit for multicolor‐barcoded multiplex RNAi at a single‐cell level

Abstract Uncovering the functions of genes in a complex biological process is fundamental for systems biology. However, currently there is no simple and reliable experimental tool available to conduct loss‐of‐function experiments for multiple genes in every possible combination in a single experiment, which is vital for parsing the interactive role of multiple genes in a given phenotype. In this study, we develop miR‐AB, a new microRNA‐based shRNA (shRNAmir) backbone for simplified, cost‐effective, and error‐proof production of shRNAmirs. After verification of its potent RNAi efficiency in vitro and in vivo, miR‐AB was integrated into a viral toolkit containing multiple eukaryotic promoters to enable its application in diverse cell types. We further engineer eight fluorescent proteins emitting wavelengths across the entire visible spectrum into this toolkit and use it to set up a multicolor‐barcoded multiplex RNAi assay where multiple genes are strongly and reliably silenced both individually and combinatorially at a single‐cell level.


27th Sep 2021 1st Editorial Decision
Dear Dr. Wang Thank you for the submission of your research manuscript to our journal. I am sorry for the delay in handling your manuscript, but we have only recently received the full set of referee reports that is copied below.
As you will see, both referees acknowledge that the multiplex reporter system will be a valuable resource for the community. However, referees 1 and 2 also point out several technical concerns and have a number of suggestions for how the study should be strengthened, and I think that all of them should be addressed. Please also provide further validation of the RNAi efficiency by at least analysing a subset of shRNAmiRs at single copy level as suggested by referee 2.
Given these constructive comments, we would like to invite you to revise your manuscript with the understanding that the referee concerns (as detailed above and in their reports) must be fully addressed and their suggestions taken on board. Please address all referee concerns in a complete point-by-point response. Acceptance of the manuscript will depend on a positive outcome of a second round of review. It is EMBO reports policy to allow a single round of revision only and acceptance or rejection of the manuscript will therefore depend on the completeness of your responses included in the next, final version of the manuscript.
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You can use this link to submit your revision: https://embor.msubmit.net/cgi-bin/main.plex Yours sincerely, Martina Rembold, PhD Senior Editor EMBO reports *********************** Referee #1: The authors describe in this manuscript a set of tools to facilitate the combinatorial gene knockdowns using microRNA-based shRNA. The main advances are a simplification in the shRNA cloning strategy and an array of fluorescent proteins added to the shmiRNA expression vector to trace combinatorial perturbations. First, the ease of cloning into the novel shmiRNA vector (miR-AB) is highlighted, while also showing the efficiency of knockdowns using said vector. Several versions of that vector (different promoters and fluorescent proteins) are then validated by showing knockdowns of a small set of genes. This panel of vectors will be a valuable resource for combinatorial knockdowns. Lastly, combinatorial knockdowns are then performed up to a maximum of four perturbations per cell. The performed experiments and presented data are technically appropriate and scientifically sound and of value to the scientific community. However, some experiments and in particular some figures would greatly benefit from some clarification to make the manuscript better understandable and underline the key messages. 1) Figure 3 and figure 5 should be clarified. First, quantifying and visualizing the level of successful knockdown will greatly improve the message of these two experiments. To do so, one could add a gating line based on the expression level in the parental cell lines -in particular for the promoter comparison. Figure 5 needs the knockdown quantification for two reasons. On one hand, having a table to summarize these levels will make the figure easier to understand. But more importantly, the table should also include the percentages of double/triple and quadruple knockdowns. This data is very important and can only be inferred from the histograms.
Minor points: 1) A baseline such as a non-stained or even better an isotype stained control should be added to cell surface marker stained flow cytometry data as a baseline representing non-expressing cells.
2) It would be nice to mention the time and cost saving of miR-AB vs miR-E in the discussion.
3) This statement in the discussion should be toned down: 'Since all miR-AB-based shRNAmirs used in this study showed potent RNAi efficacy, miR-AB, in combination of SplashRNA or shERWOOD, would outcompete most of the current RNAi tools which always need a pilot experiment to validate their candidate shRNAs.' This statement appears premature given the limited number of shmiRNA assessed here. A more comprehensive analysis (eg. targeting essential genes with multiple shmiRNA targeting each gene and confirmation of the efficiency of knockdown of this larger panel) would be needed to state that shmiRNA is superior to previously reported strategies.
4) The section in the discussion on fluorescent protein compatibility should be streamlined and clarified. Together with the informative tables with recommendations on panel design, this section can be kept much simpler and less confusing. 5) Several typos should be corrected. See for example: Page3 -Dice instead of Dicer Page 5 -packaging instead of packaged Page 7-picogram instead of picograms Figure 1B -Transform instead of transfect 293T cells Throughout the manuscript, the authors sometime switch between first person (I) and third person (we).
Referee #2: 1. Summary Wang et al. develop "miR-AB", a novel shRNAmiR-system based on the human miR30A backbone. In miR-AB, restriction sites are introduced into the lower stem region which facilitates cloning since annealed oligos can be used directly for cloning into the miR-AB backbone, thereby by omitting laborious and expensive PCR-amplification of oligos. The authors develop the system into a versatile toolkit and show that miR-AB works as polycistronic construct after different fluorescent proteins and can be driven by several, widely used eukaryotic promoters. The manuscript has merits, especially regarding the multicolor-barcording RNAi. However, I have some reservations about the novelty and technical execution of parts of the study that should be addressed before a potential publication.
2. Key results miR-AB is a versatile and easy to use RNAi trigger in different contexts.

Clarity and context
The manuscript is clearly written and the goals are clear. The reader can follow the course of analysis and conclusions drawn from the data.

Results: "De novo cloning of shRNAmir into miR-AB is simple, inexpensive and error-proof"
In this part the authors describe the development of miR-AB. miR-AB was obtained by introducing ApaI/BamHI restriction sites into the lower stem and bulge of the widely used miR30-E backbone. By moving the restriction sites closer together, it is possible to clone directly two annealed oligos of 75 bp and 65 bp length into the backbone, whereas in the classical miR30-E system (Fellmann/Zuber 2013) the oligos needed are longer (97mers) and have to be amplified by PCR before cloning into the miR30-E backbone. However, this strategy to rapidly clone shRNAmiRs is not novel. Several groups, e.g. Adams et al. (An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries Biomaterials 2017) have developed similar systems, albeit they mostly use BsmBI (Type IIS) restriction sites. The authors claim that these systems suffer from low cloning efficacy but do not provide data to support this claim. A head-to-head comparison between these systems and miR30-AB has not been performed. Given that complex shRNAmiR librarys have been cloned and validated by NGS with these systems, efficacy and accuracy does not seem to be a bottleneck there. At least, it should be mentioned that similar systems have been developed before and appropriately referenced. Claims about the superiority of miR30-AB should be deleted when there is no data to support them.

Results: "miR-AB shows outstanding RNAi efficiency in vitro"
In this paragraph, the knockdown efficacy is compared between the novel miR30-AB and the miR30-E shRNAmiR system for different shRNAs predicted by SplashRNA. However, I have great concerns about the validity of the results of this paragraph, since the authors have performed the knockdown experiments in cell lines where "A nearly 100% of transduction efficiency was achieved after 72h of infection". This means, the authors worked with cell lines had multiple, possibly tens or even hundreds of integrations of the viruses encoding the shRNAmiRs. Since maximal 100% of the cells can be green after transduction it remains unclear whether cells harboring a comparable number of integrations and therefore vector dosage and shRNAmiR payload were compared in these experiments. It is nowhere mentioned at which MOI the cells were transduced. The efficacy of RNAi triggers must generally be compared at singly copy level (Fellmann et al. Cell Reports 2013), this means a maximum of 30% of transduced cells is allowed. To rescue this experiment the authors could determine the vector copy number in the samples or at least state at which MOI the cells where transduced. However, at least three independent shRNAmiRs should be evaluated at single copy level (not necessarily in all cell lines) to support the conclusion that miR30-AB is non-inferior to miR30-E. SplashRNA predicts very powerful RNAi-triggers, which can be difficult to compare due to their high knockdown potency. E.g. the one of the most powerful shRNAs Pten.1523 performs well even when expressed from the original miR-30 backbone. Therefore I would suggest to include some medium-power shRNAmiRs, such as Pten.1524 or Pten.932 (mouse -use NIH3T3) to compare knockdown potency in RNAi triggers that have the potential to improve or deteriorate when expressed from different miR30 backbones.

Results
: "Multiple eukaryotic promoters guarantee miR-AB-based RNAi in various cell types" "These new constructs were packaging in 293T cells as effectively as the original vector with the human CMV promoter and maintained the intensity of the Venus reporter." I presume the authors wish to state that viral titers are the same for the constructs with different promoters. If this is the case data to support this notion should be shown. The FACS plots in Figure 3 should be supported by the MFIs in a supplementary table to support the conclusions drawn in this paragraph.

Results
: "Construction of a multi-promoter and multicolor miR-AB-based viral toolkit" "These fluorescent protein-carrying miR-AB viral vectors can produce high viral titers and generate" -the titers achieved with these constructs on average should be mentioned to support this notion. Second, it would be important to include cell viability assays to show whether the expression of multiple shRNAs and fluorescence proteins is toxic. To determine even subtle effects I would suggest to transduce cells with the 4 different vectors (Azurite, EGFP, Ametrine, mCherry) each harboring a neutral shRNA and follow them over time in a bulk culture to see whether cells harboring more FPs are outdiluted slowly over time. It is important to show this stability over time since it can greatly impact in vivo experiments.

Our response:
We are very pleased that you are interested in the study and are thankful for your helpful comments. We revised the main text and Figures as you suggested. Moreover, we did new experiments to show miR-AB's knockdown efficiency at single copy level ( Figure 2E, 2F) and multiple shRNAmir transduction is not cytotoxic ( Figure 4F). Furthermore, we developed new vectors for easy test of the activity of the promoters used in this toolkit ( Figure 4D). According to the format requirement of EMBO reports, the Results and Discussion were combined and the text relating to the new data or being dramatically revised were highlighted for your easy recognition. 1) Figure 3 and figure 5 should be clarified. First, quantifying and visualizing the level of successful knockdown will greatly improve the message of these two experiments. To do so, one could add a gating line based on the expression level in the parental cell lines -in particular for the promoter comparison. Figure 5 needs the knockdown quantification for two reasons. On one hand, having a table to summarize these levels will make the figure easier to understand. But more importantly, the table should also include the percentages of double/triple and quadruple knockdowns. This data is very important and can only be inferred from the histograms.

Our response:
As you suggested, Figure 3 was quantified by showing the MFI of APC-FAS (bottom panels). Figure 5 and Figure EV1 were also quantified by showing the MFI of target genes ( Figure EV2). The percentages of all single, double, triple and quadruple knockdown cells were included in Figure EV2. "The overall percentage of these populations was >50% (untransduced cell percentage was <50%, top bars in Fig EV2), indicating these viruses had high viral titers because primary CD8+ T cells are harder to be infected than cell lines. The lowest individual percentage of these population was >1%, indicating there are enough cells for FACS analysis of each population in this 13th Dec 2021 1st Authors' Response to Reviewers assay (hundreds of cells can give a clear FACS population)." have been included in the text.
As another reviewer suggested, we used MFI but not gating line to quantify the expression of target genes, because these cells are homogeneous, and all cell lines express FAS (Figure 3) and all activated CD8+ T cells express CD127, CD44, CD90 and CD8 (Figure 5 and EV1). A gating line is commonly used for separating positive population from negative population in a heterogeneous cell population. If we look at the histograms of the control shRNA and FAS shRNA in Figure 3, they both have only one peak. Moreover, the FAS shRNA peak locates on the left of control shRNA peak, indicating all cells express FAS. If some cells are FAS negative in the control shRNA peak, the peak of FAS shRNA should be narrower and not closer to the left than control shRNA peak.

Minor points:
1) A baseline such as a non-stained or even better an isotype stained control should be added to cell surface marker stained flow cytometry data as a baseline representing non-expressing cells.

Our response:
To show RNAi effects in our FACS experiments, we used the commonly used method in immunology, i.e. comparison between control shRNA-transduced cells and target gene specific shRNA-transduced cells after staining with same target protein-specific antibody. We didn't use isotype control since it is hard to get the basal expression level from it. An ideal isotype control should guarantee same species, same heavy chain and light chain class, same fluorochrome and same fluochrome:antibody ratio. This is almost impossible. "Same" isotype controls from different company or different batches from same company always give different results. Therefore, some scientists argue, even abandon the use of isotype controls in some experiments. If we go to Purdue Cytometry List, the best cytometry forum, we will see lots of arguments relating to isotype controls. In my opinion, Isotype controls are most useful when a new fluochrome-conjugated antibody is developed and needs to be validated. In this study, we used reliable fluorophore conjugated monoclonal antibodies that have been wildly used for long time.
To test the knockdown efficiency of endogenous gene, the best way is to use target gene knockout cells as negative control. After staining of knockout cells and the cells to be analyzed with the same target gene-specific antibody, we can get accurate information of the target gene expression changes. However, knockout cells are not always available for some experiment.
The purpose of FACS experiments in this study is to show the differential promoter activity (Figure 3) or the feasibility of carrying out a multicolor-based multiplex RNAi (Figure 5 and EV1). The exact RNAi efficiency has been shown in the western blot data and GFP RNAi FACS data in Figure 2. The GFP RNAi FACS data clearly showed both miR-AB and miR-E backbone mediated strong RNAi by Sh2 and Sh3 (very few low-MFI GFP+ cells left).
2) It would be nice to mention the time and cost saving of miR-AB vs miR-E in the discussion.

Our response:
The time and cost saving of miR-AB vs miR-E have been included in the text as follows: "This new approach significantly reduces the cost and time in production of shRNAmir. Specifically, cloning of a shRNAmir by miR-AB only costs the synthesis of 142bp oligos (75bp + 67bp), takes 0.5 hour for annealing of miR-AB oligos and requires minimal labor (mixing and pipetting the oligos), while its cloning by PCR is more cost-ineffective and labor-intensive, including the synthesis of 97bp oligo, running a PCR, separating PCR product on agarose gel, gel purification of PCR product and restriction enzyme digestion of PCR product followed by purification and quantification."

3) This statement in the discussion should be toned down: 'Since all miR-AB-based shRNAmirs used in this study showed potent RNAi efficacy, miR-AB, in combination of SplashRNA or shERWOOD, would outcompete most of the current RNAi tools which always need a pilot experiment to validate their candidate shRNAs.
This statement appears premature given the limited number of shmiRNA assessed here. A more comprehensive analysis (eg. targeting essential genes with multiple shmiRNA targeting each gene and confirmation of the efficiency of knockdown of this larger panel) would be needed to state that shmiRNA is superior to previously reported strategies.

Our response:
Your suggestion is pretty accurate because we didn't make a comprehensive comparison of shRNAmir vs shRNA. Therefore, this sentence has been removed.

Our response:
As you suggested, the text was revised and Figure EV3 was made to clarify the compatibility of these fluorescent proteins. To put it in a simple way, we highlighted in Figure EV3 "Don't use Azurite and mTagBFP2 together, or GFP and Venus together unless an advanced flow cytometer or microscope is available. Any other combinations among these eight fluorescent reporters can be used in most commercial flow cytometers and fluorescent microscopes". In Figure EV3, a detailed protocol of setting up a multicolor assay was included and some suggestions and recommendations were given for avoiding mistakes.

Our response:
Thank you for your pointing out the mistakes. We really appreciate. All typos have been corrected.

Our response:
We are very thankful for your valuable comments and suggestions. We did new experiments and revised the text and data according to your and other reviewer's suggestions. Furthermore, we developed new vectors for easy test of the activity of the promoters used in this toolkit ( Figure 4D). According to the format requirement of EMBO reports, the Results and Discussion were combined and the text relating to the new data or being dramatically revised were highlighted for your easy recognition.

Clarity and context
The manuscript is clearly written and the goals are clear. The reader can follow the course of analysis and conclusions drawn from the data.

In this part the authors describe the development of miR-AB. miR-AB was obtained by introducing ApaI/BamHI restriction sites into the lower stem and bulge of the widely used miR30-E backbone. By moving the restriction sites closer together, it is possible to clone directly two annealed oligos of 75 bp and 65 bp length into the backbone, whereas in the classical miR30-E system (Fellmann/Zuber 2013) the oligos needed are longer (97mers) and have to be amplified by PCR before cloning into the miR30-E backbone. However, this strategy to rapidly clone shRNAmiRs is not novel. Several groups, e.g. Adams et al. (An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries Biomaterials 2017) have developed similar systems, albeit they mostly use BsmBI (Type IIS) restriction sites. The authors claim that these systems suffer from low cloning efficacy but do not provide data to support this claim. A head-to-head comparison between these systems and miR30-AB has not been performed. Given that complex shRNAmiR librarys have been cloned and validated by NGS with these systems, efficacy and accuracy does not seem to be a bottleneck there.
At least, it should be mentioned that similar systems have been developed before and appropriately referenced. Claims about the superiority of miR30-AB should be deleted when there is no data to support them.

Our response:
As you suggested, the BsmBI-based paper was referenced. Since we didn't make a head-to-head comparison between miR-AB and BsmBI-based backbone, we don't know if their cloning efficacy and reliability are similar or different. Our miR-AB based shRNAmir cloning doesn't need oligo phosphorylation, which is an advantage over BsmBI-based backbone. Moreover, BsmBI (3.7$/ul, NEB) is more expensive than BamHI (0.144$/ul, NEB) and ApaI (0.62$/ul, NEB). These words "simple, inexpensive and error-proof" are only a description of our data in Figure 1, not a claim that miR-AB is superior to any other approaches. Any other words or phrases that might remind audience of miR-AB's superiority have been removed. The aim of these study is to develop a RNAi toolkit that can be used to set up a multicolor-based multiplex RNAi assay. These miR-AB vectors can provide a choice for RNAi users.

Results: "miR-AB shows outstanding RNAi efficiency in vitro"
In this paragraph, the knockdown efficacy is compared between the novel miR30-AB and the miR30-

Our response:
Your comments and suggestions are pretty instructive and meaningful. As you suggested, we tested Pten.1524 and Pten.932 RNAi efficiency in the context of less than 30% transduction efficiency. After FACS sorting of GFP+ 3T3 cells, we did western blot to determine the expression level of Pten ( Figure 2E). Consistent with the miR-E paper, both Pten.1524 and Pten.932 resulted in a significant reduction of Pten protein level at low transduction efficiency. These potent shRNAmirs displayed no significant knockdown difference in the context of miR-AB vs. miR-E.
To more intuitively show miR-AB's RNAi efficiency at single copy level, we carried out a flow cytometry-based experiment to show transduction efficiency and RNAi efficiency simultaneously. To this end, we cloned three shRNAmirs targeting the GFP reporter in a lentiviral vector with miR-AB or miR-E backbone and analysed their RNAi potency after transduction of MC38 or 293T cells. AS shown in Fig 2F, in the context of less than 30% transduction efficiency, these shRNAmirs showed potent (Sh1 and Sh2) or moderate (Sh3) knockdown efficiency (indicated by *) in GFP+ cells. Their RNAi potency was comparable between in the miR-AB vs. miR-E backbone (indicated by NA). It should be noted that the medium-power Sh3 data is more meaningful to compare miR-AB vs. miR-E than Sh1 and Sh2, because GFP was almost undetectable in Sh1 and Sh2 transduced cells (their MFI only represents the leftover GFP+ cells after RNAi). This paragraph has been included in the text.

Results: "Multiple eukaryotic promoters guarantee miR-AB-based RNAi in various cell types"
"These new constructs were packaging in 293T cells as effectively as the original vector with the human CMV promoter and maintained the intensity of the Venus reporter." I presume the authors wish to state that viral titers are the same for the constructs with different promoters. If this is the case data to support this notion should be shown. The FACS plots in Figure 3 should be supported by the MFIs in a supplementary table to support the conclusions drawn in this paragraph.

Our response:
The titers of different promoter-based lentivirus were shown in the text as follows "A cost-effective transfection of 293T cells with these constructs by PEI (~40% transfection efficiency) can generate 3-5 × 10 6 transduction unit titers of virus from one well of sixwell plate. Commercial transfection reagent can generate higher titers by increasing transfection efficiency." As you and another reviewer suggested, Figure 3, Figure 5 and Figure EV1 were quantified by showing the MFI of target proteins.

Results: "Construction of a multi-promoter and multicolor miR-AB-based viral toolkit"
"These fluorescent protein-carrying miR-AB viral vectors can produce high viral titers and generate" -the titers achieved with these constructs on average should be mentioned to support this notion.

Our response:
In this study, we mainly used retrovirus carrying different fluorescent proteins to infect primary mouse CD8+ T cells (Figure 4, 5 and EV1), which are harder to be infected than cell lines. Immunologists always use undiluted retrovirus to infect primary CD8+ T cells for short time (4h in this study) because even though highly-concentrated virus was used, the transduction efficiency would not be higher than 80-90%. As shown in our multicolor assay using multiple retrovirus carrying different fluorescent reporters at 1:1 ratio ( Figure S4), more than 50% of cells were infected by unconcentrated virus (untransduced cell percentage is <50%), indicating these retroviral vectors can produce high viral titers. Moreover, each FACS plot in Figure 4E clearly showed two fluorescent reporter single positive cell populations (top left quadrant and bottom right quadrant) have similar percentage, indicating all the viral vectors were packaged efficiently. Our recent retrovirus titering assay using CD8+ T cells showed one well of 6-well plate by PEI transfection produced ~0.5-1 ×10 6 transfection unit of virus. To clarify, "The overall percentage of these populations was >50% (untransduced cell percentage was <50%, top bars in Fig EV2), indicating these viruses had high viral titers because primary CD8+ T cells are harder to be infected than cell lines. The lowest individual percentage of these population was >1%, indicating there are enough cells for FACS analysis of each population in this assay (hundreds of cells can give a clear FACS population)." was added in the main text.
As you suggested, we transduced 293T cells with four lentiviral vectors (Azurite, GFP, Ametrine or mOrange as reporters) harboring shCD4 or shCD19 and tested the reporter-positive cells expansion over time (from day 4 to day 22). As shown in Figure  4F, we didn't observe obvious difference of cell expansion between untransduced, single fluorescent reporter-transduced and four fluorescent reporter-transduced subsets at the given time point. Accordingly, we added a new paragraph as follows "The aim of this study was to develop a novel multiplex RNAi assay. We chose shRNAmir but not conventional shRNA in this assay because shRNAmir displayed minimal cytotoxicity. Indeed, our experimental data showed the co-transduction of 293T cells with four viruses carrying a neutral shRNAmir did not significantly alter the four fluorescent reporters-expressing cell expansion after 22 days of culture ( Fig 4F). Since the multiple shRNAmirs-expressing cells always account for a small portion of whole cell population (<10% in this experiment), like low efficiency transduction of a single shRNAmir, it is very likely that they express each shRNAmir at single copy level. So, their total shRNAmir level will not be super abundant. Given the advantage of shRNAmir over conventional shRNA in maintaining cell homeostasis, multiple shRNAmir transduction might not be problematic." Thank you for the submission of your revised manuscript to EMBO reports. We have now received the full set of referee reports that is copied below.
As you will see, all referees are very positive about the study and request only minor changes to clarify text and figures. Please address the remaining concerns from referee 2.
From the editorial side, there are also a few things that we need before we can proceed with the official acceptance of your study.
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-Please also add a header for the Expanded View Figure legends -During our routine image and figure analysis we noted that all Western blots have rather high contrast settings. Please reduce the contrast and I also recommend providing the source data for the Western blots.
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Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
Sample size was chosen according to most relevant studies. graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner. a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.

definitions of statistical methods and measures:
a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).

Reporting Checklist For Life Sciences Articles (Rev. June 2017)
This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. These guidelines are consistent with the Principles and Guidelines for Reporting Preclinical Research issued by the NIH in 2014. Please follow the journal's authorship guidelines in preparing your manuscript.