V1.

0 October 2022

Author instructions
• Please fill in the template below.
• Replace any text within [square brackets] with text relevant for your protocol.
• Brief instructions are in italics. Additional instructions are highlighted and appear in Commented [HLY1]: This is an example of what additional
instructions will look like.
the comment boxes, which can be found when you choose “All Markup” in the Track
Changes menu.
• Please do not change any section headings (e.g., Before you begin, Key resources
table).
• Before submitting your protocol, please delete the instructions on this page as well Commented [HLY2]: You can see an example of a submitted
protocol as a Word document here.
as all italicized instructions and all comments in this template.
• Please submit your manuscript to
https://www.editorialmanager.com/heliyon/default2.aspx . Be sure to upload the
Word version of this manuscript at submission.
• You can include figures within this document or upload the figures separately.
• If your protocol contains code, please follow these guidelines:
o For small snippets of code, please use a monospace font and indented text as
shown below:
>save.image("Heliyon.Rdata")

o For larger code segments, please put the code in an in-line text box in Word
exactly where the code should appear (see example below). Please consult this
walk through on how to add in-line text box.
>if(!require(devtools)){
> install.packages("devtools") # If not already installed
>}
>devtools::install_github('protocols')
[Title]
Choose a concise title that includes the name of the method/technique, the purpose of the protocol,
and the model system or organism.

[Author (First Name Surname),1,2,3,* Author (First Name Surname),1,2 and Author (First Name Surname)1,4,** Commented [HLY3]: Please list author names as First Name
Surname.
1
Affiliation, including city, state, postal code, and country
2
Optional footnotes such as current addresses or equal-contribution notes
3
Technical contact
4
Lead contact Commented [HLY4]: Please include email addresses for bot
the lead and technical contact.
*Correspondence: Author’s email The lead contact is the long-term contact for resources related t
**Correspondence: Author’s email] the paper and must match the name/contact in the Resource
availability statement.
The technical contact is the point person for technical questions
about the protocol.

Summary
[The summary is a paragraph no longer than 80 words. Any background information should be limited
to one sentence. The summary should focus on the major steps of the protocol and should highlight
what the protocol does. You can also include applications or limitations of the protocol.]

For complete details on the use and execution of this protocol, please refer to X et al. (1). Commented [HLY5]: If your protocol was derived from a
paper that has already been published, this line is mandatory
and does not count towards the word limit.

Graphical abstract
The GA is a graphical description of the protocol that highlights the time for each major step. Click
here to see examples. We encourage you to submit a GA with your initial submission, but it is not
required.

Before you begin Commented [HLY6]: Editor tip:

Describe the protocol specifically for one particular example.
You can describe here how else you have used the protocol.

Describe what needs to be set up or prepared before a researcher begins the protocol. Please use For example,
“The protocol below describes the specific steps for using HeLa
numbering (up to three levels: 1,2,3; a,b,c; i,ii,iii) to list steps. Do not use bullet points in this section. cells. However, we have also used this protocol in HEK293 cells
and NIH3T3 cells.”

Institutional permissions (if applicable) Commented [HLY7]: Editor tip:

Use the active voice and the present tense throughout the
[Any experiments on live vertebrates or higher invertebrates must be performed in accordance with protocol, e.g., Culture HeLa cells for 2 days.
relevant institutional and national guidelines and regulations. In the before you begin section, a
statement identifying the committee approving the experiments and confirming that all experiments
conform to the relevant regulatory standards must be included.
In addition, you should provide a statement reminding readers that they will need to acquire
permissions from the relevant institutions].

[Preparation one] Commented [HLY8]: Replace this description with your own
text, e.g., design primers, culture cell lines.

Timing: [s, min, h, days, or weeks] Commented [HLY9]: Timing must be included with each
major step. It is also possible to include additional timing notes
for sub steps, but only if the total time for the major step is
1. [First step of preparation one. Use numbers to order steps.] included.
a. [Sub-steps under first step. Use letters for ordering of sub-steps.]
b. [Sub-step two, as needed.]
i. [Third-level sub-steps under second-level sub-steps. Use lowercase Roman
numerals for ordering.]
ii. [Third-level sub-step two, as needed.]
2. [Second step of preparation one]

[Preparation two]

Timing: [s, min, h, days, or weeks]

3. [First step of preparation two. Use numbers to order steps.] Commented [HLY10]: Note that the step numbers continue
from “Preparation one”.
a. [Use letters for ordering of sub-steps.]
b. [Sub-step two, as needed.]
4. [Second step of preparation two]

CRITICAL:[when applicable, note steps that are critical for success of the protocol] Commented [HLY11]: Editor tip:
Include figures and/or videos to help guide critical steps.
Include figure callouts in the text (e.g., Figure 1) to indicate
where figures should appear.

Key resources table Commented [HLY12]: Do not change the headings in the key
resources table. Please note that the heading "Other" is for all
equipment and items not covered by other headings.
The key resources table (KRT) highlights key reagents, resources, and equipment used in the protocol.
Include vendor/manufacturer and the model/catalog number for each reagent or resource. For
reagents/resources that are critical for the success of this protocol, please provide links to the
corresponding vendor sites. To create the key resources table using Word, please use this table
template (http://www.cell.com/pb-assets/journals/research/cell/methods/table-template1.docx).
Although the KRT template states that the table should be uploaded as a separate document, it is also
acceptable to include the KRT in the main text file. However, please include only one copy of the
KRT—either in the main text file or uploaded as a separate file.
Materials and equipment (optional)
Here, you can provide additional information regarding equipment setup, details about custom
software used, and notes on alternative materials and equipment. This optional section is a
complement to the key resources table. All recipes should be in this section; these tables do not require
legends or call outs and can stay in this section. Please use bullet points (up to three level) to list
items. Do not use numbering (1,2,3; a,b,c; i,ii,iii) in this section.

Note: recipes with ≥3 ingredients should be presented as tables.

[Name of solution/buffer/medium]
Reagent Final concentration Amount
Component A (100 µM) X 1 mL
Component B (100 µM) Y 1 mL
10 × ligase buffer Z 5 mL
T4 polynucleotide kinase Z 1 mL
ddH2O n/a 42 mL
Total n/a 50 mL
[Note on storage conditions: temperature, maximum storage time]

Note: short recipes should be presented as a bulleted list.

• 5% Compound X solution: add 5 g compound X in 100 mL ddH2O.
[Note on storage conditions: temperature, maximum storage time]
• Media supplemented with X: DMEM with 5% of X
[Note on storage conditions: temperature, maximum storage time]

CRITICAL: [Mark any chemicals or reagents that are harmful or toxic with a brief explanation of the
hazard, and state clearly how to take precautions when handling these agents.]

Alternatives: [Mention any known alternative materials or equipment in this format.]

Step-by-step method details

This section lists the major steps and provides step-by-step details and timing for each major step.
Please use continuous number for this section. Please use numbering (up to three levels: 1,2,3; a,b,c;
i,ii,iii) to list steps. Do not use bullet points in this section.

[Your major step one] Commented [HLY13]: Replace this description with your
own text, for example: Transfection of cell line; Purification of
protein from bacteria.
Timing: [s, min, h, days, or weeks] Commented [HLY14]: Timing must be included with each
major step. It is also possible to include additional timing notes
for sub steps, but only if the total time for the major step is
included.
[Include a brief description about what this major step accomplishes; this will help other researchers Commented [HLY15]: Editor tip:
For steps using manufacturer's protocols, please include link(s)
repeat and troubleshoot the protocol.] to the protocols.

1. [First step of major step one. Use numbers to order steps.]

a. [Sub-steps under first step. Use letters for ordering of sub-steps.]
b. [Sub-step two, as needed.]
i. [Third-level sub-steps under second-level sub-steps. Use Roman numerals for
ordering.]
ii. [Third-level sub-step two, as needed.]
2. [Second step of major step one]

CRITICAL: [Particularly important instructions or factors should be called out in this format. Please
provide figures or videos for guiding critical steps. Click here to see examples of useful figures and Commented [HLY16]: Editor tip:
Smart phone camera videos are usually adequate.
here for example videos.] Use landscape not portrait view.
Video files should be less than 50MB and be in .MP4 format.

[Your major step two]

Timing: [s, min, h, days, or weeks]

[Include a brief description about what this major step accomplishes. This will help other researchers
repeat and troubleshoot the protocol.]

3. [First step of major step two. Use numbers to order steps.] Commented [HLY17]: Editor tip:
Step numbers continue from “Your major step one”.
a. [Sub-steps under first step. Use letters for ordering of sub-steps.]
b. [Sub-step two, as needed.]
4. [Second step of major step two] Troubleshooting 1 Commented [HLY18]: Editor tip:
If relevant, include links between the troubleshooting section
and the major steps.
Optional: [Call out optional steps in this format; optional steps are not numbered.]

Note: [Please include general notes or explanations as a note. Notes are not numbered and should be
near the relevant step.]

Pause point: [If relevant, list potential pause points with time intervals.]

Note: Present PCR reaction master mix with the following format (if relevant)

PCR reaction master mix

Reagent Amount
DNA template
DNA Polymerase
Primer 1
Primer 2
 Buffer x
Buffer y
ddH2O

Note: Present PCR reactions with the following format (if relevant)

PCR cycling conditions

Steps Temperature Time Cycles
Initial Denaturation 98 °C 30 sec 1
Denaturation 98 °C 10 sec
Annealing 55 °C 20 sec 25-35 cycles
Extension 72 °C 10 min
Final extension 72 °C X min 1
Hold 4 °C forever

Expected outcomes
Include information about the anticipated outcomes of the protocol, e.g., estimated yield of DNA
extraction, images of protein expression pattern. We encourage authors to provide figures to illustrate Commented [HLY19]: Editor tip:
It is acceptable to reuse parts of figures from your previous
the expected outcomes (see examples here.) and describe the expected results in paragraphs. research articles in your protocol. However, please be sure to
obtain permission and include that information in the relevant
figure legend.
Quantification and statistical analysis (optional) Elsevier (including Cell Press) authors retain the right to use or
re-use portions or excerpts in other works (see
https://www.elsevier.com/about/our-
Please provide details about the methods used for data processing, quantification, and statistical business/policies/copyright/personal-use).

analysis. Include criteria for data inclusion/exclusion. If the protocol outcome requires a complex
statistical or computational analysis, include a sample data set to allow others to repeat your
approach, and provide instruction on the interpretation of the raw data (Table 1). Either numbering
(up to three levels: 1,2,3; a,b,c; i,ii,iii) or bullet points (up to three levels) can be used to list steps in this
section.

Commented [HLY20]: Editor tip:

Table 1. [Example of raw data measured on a plate reader] Include a title for each table.
Include table callouts in the text close to where it is first cited
Time Temp. Raw data (e.g., Table 1).
Prepare all tables using the table function in a word-processing
- - Blank Blank Avg. blank Negative Negative Target Target program.
control control Do not include colored text, indented text, shading, or panels in
the table.
Please ensure that all table and cell lines and borders are visible
 - - A1 A2 - A3 A4 A5 A6

0:00:00 32 °C X1 X2 X3 X4 X5 X6 X7

00:10:00 32 °C Y1 Y2 Y3 Y4 Y5 Y6 Y7

00:20:00 32 °C Z1 Z2 Z3 Z4 Z5 Z6 Z7

Limitations
[Describe possible limitations of the protocol, including any situations where the protocol may be
unreliable or unsuccessful. Describe environmental factors or mechanical limitations that might affect
the validity of the results.]

Troubleshooting Commented [HLY21]: Editor tip:

In the troubleshooting section, please refer to the correspondin
protocol step; creating a hyperlink to the step improves
Describe any problems that might arise from running the protocol and provide possible solutions. readability.

Make sure to flag them in the corresponding steps of the protocol and refer to this section. If
applicable, this section can also be the space to include information about alternates, i.e., what
reagents and/or equipment has some flexibility and what cannot be changed. If you wish to include a
list for each problem/potential solution, please use bullet points (up to three level) make a list. Do not
use numbering (1,2,3; a,b,c; i,ii,iii) in this section.

Problem 1:

[When illustrating problems, we encourage the use of figures/videos to indicate a good outcome
versus a bad outcome. See examples here and here. Please mention the related step in the protocol,
if relevant (related to Step 4).]

Potential solution:

• [Describe the details of the solution to resolve this problem.

• You can use a bulleted list if needed.]

Resource availability
We require three subheadings in this section (lead contact, materials availability, and data and code
availability).
Lead contact Commented [HLY22]: Editor tip:
The lead contact should match the cover page. Do not include a
Further information and requests for resources and reagents should be directed to and will be fulfilled technical contact here.
by the lead contact, [lead contact’s name] (lead contact’s email).

Materials availability Commented [HLY23]: Materials availability statement

examples:
Provide information regarding availability of newly generated materials associated with this protocol, • Plasmids generated in this study have been deposited to
including any conditions for access. [Addgene, name and catalog number or unique identifier].
• Mouse lines generated in this study have been deposited to
[the Knockout Mouse Project (KOMP), name and catalog numbe
Data and code availability or unique identifier].
State whether the protocol includes all datasets generated or analyzed during this study. Provide • This study did not generate new unique reagents.
• There are restrictions to the availability of [reagent] due to
information about data and code availability. [reason why restrictions exist].
Commented [HLY24]: Data and code availability
Acknowledgments statement examples: (Statements with multiple types of
datasets may use a combination of statements)
• The [datasets/code] generated during this study are available
[Enter the following information here: 1) all funding sources; 2) collaborators and/or core facilities at [name of repository]: [accession code/web link].
• The published article includes all [datasets/code] generated o
contributing to the work.] analyzed during this study.
• The [datasets/code] supporting the current study have not
been deposited in a public repository because [reason why data
are not public] but are available from the corresponding author
Author contributions on request.
• This study did not generate/analyze [datasets/code].
• There are restrictions to the availability of [dataset/code] due
[Mention each individual author with a statement outlining the contribution of each author to the to [reason why restrictions exist].
• Original/source data for [figures/datatype] in the paper is
work.] available [e.g., Mendeley Data DOI].

Declaration of interests
[Please disclose competing interests for all submitted content. Include a “declaration of interests”
section in the text of all articles even if there are no interests declared.]

References
Please format all in-text citations as superscripted numbers (e.g., “Multiple reports support this
observation.1,2”).

Figure legends Commented [HLY25]: Editor tip:

At initial submission, you can include your figures in the text or
as a separate document.
Figure 1: [Include a clear and concise figure title for each figure. Figure legends are suggested, but not
required.]
Methods Video S1: [Include a clear and concise title for each video. Be sure to mention the relevant
step, e.g., Preparing microscope slides, related to step 3.]