Verified Rigor RecordRigor Reviewed

Rigor-Reviewed Manuscript

Kinetic organization of the genome revealed by ultraresolution multiscale live imaging.

Name: Verified Rigor Record — Kinetic organization of the genome revealed by ultraresolution multiscale live imaging.
Item: Kinetic organization of the genome revealed by ultraresolution multiscale live imaging.
Rating: 81
Author: Αlpha¹

Lee J, Chen LF, Gaudin S, Gupta K, Novacic A, Spakowitz A, Boettiger AN.

Paper source

Open DOI

Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 5, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Observational Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: moderateReviewer agreement: lowEngine: 7.5.0Source: full text

Not verified in this report

References were not verified against Crossref/OpenAlex.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper presents a technically impressive live-imaging platform and provides novel insights into chromatin dynamics. The main methodological weaknesses are incomplete reporting of study design rigor elements (blinding, power analysis, inclusion/exclusion, outlier handling, replicate definition) and lack of key resource identifiers (cell authentication, reagent catalog numbers). Data/code sharing and statistical reporting are strengths.

Evaluation used three independent rigor reviewers (all AI) plus a copyedit pass and verification components (2 statistics checks, 1 reproducibility link, zero citation issues). The reviewers diverged on ethical approvals (applicability of IACUC), study design (applicability of randomization/blinding), and key resources (stringency of reagent identification); the synthesized judgments weigh the combined evidence. Not applicable dimensions were excluded from scoring.

12 major claims checked against the paper's own evidence: all adequately supported.

Recomputed 2 tests: 2 consistent, 0 inconsistent, 2 via agent-written checks.

1 integrity concern flagged (0 high).

Rigor

Eight Dimensions4 pass · 2 warn · 1 fail

12 major claims4 partial8 supported

Prior work is thoroughly cited, including Hi-C, loop extrusion models, and live imaging studies. The rationale linking prior knowledge to the study's objectives is logical and explicit. The paper directly addresses how limitations of prior approaches (resolution, coverage, throughput) are overcome by the new technology.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The paper establishes a strong scientific premise by extensively citing prior work on genome organization and loop extrusion, clearly identifying gaps in understanding dynamics, and describing how TRACK-IT addresses these gaps.

Evidence

direct quote[Introduction, paragraphs 1-3]

“However, while the contribution of these different mechanisms to the steady-state, population-averaged features has been analyzed with simulations, Hi-C sequencing, and microscopy, our understanding of the dynamics and the timescale of genome motion as a function of sequence, distance, and chromatin state is just beginning.”

direct quote[Introduction, final paragraph]

“Here we introduce TRansposon Assisted Chromatin Kinetic Imaging Technology (TRACK-IT), which allows us to measure chromatin motion in ultra-resolution in space and time, with distributed labels across a single chromosome.”

direct quote[Abstract]

“However, tools to directly observe this motion in vivo have been limited in coverage and resolution.”

PASSReviewer 2· 95% conf

The paper thoroughly cites prior work on genome organization, loop extrusion, and live imaging, establishes a clear rationale for its approach, and addresses limitations of earlier methods.

Evidence

paraphrase[Introduction, paragraphs 1–6]

“The introduction cites numerous papers on Hi-C, polymer models, loop extrusion, cohesin, live-cell imaging, and TADs.”

direct quote[Introduction, paragraph 3]

direct quote[Introduction, paragraph 5]

“Our ability to test these results across genomic scales and distinct chromatin contexts has been limited by the difficulty of labeling large numbers of precisely mapped genomic regions. The ability to detect distinct molecular forces affecting the motion of particular genome sequences has also been limited by the spatial-temporal resolution of current approaches.”

PASSReviewer 3· 95% conf

The scientific premise is well-established through extensive citation of prior work on genome organization, polymer physics, and loop extrusion, with a clear rationale linking observed gaps in dynamic understanding to the study's objectives.

Evidence

direct quote[Introduction, paragraphs 1-4]

“The mammalian genome is spatially organized in the nucleus, with complex, sequence-specific and cell-type-specific, contact patterns that correlate with transcriptional regulation, replication and repair (–). Recent single-cell DNA-tracing microscopy has shown that these characteristic patterns, as seen in contact-frequency heatmaps, are not structures that exist in folded genomes of individual cells, but statistical patterns that arise from averaging of many diverse folds from the many cells of the population ().”

direct quote[Introduction, paragraph 4]

“Our ability to test these results across genomic scales and distinct chromatin contexts has been limited by the difficulty of labeling large numbers of precisely mapped genomic regions.”

direct quote[Introduction, paragraph 5]

The study is primarily observational (live imaging of chromatin motion) but includes a controlled depletion experiment (cohesin removal via dTAG-13). No randomization method or unit is reported (not applicable for observational imaging). Blinding is not mentioned (not reported for image analysis). Power analysis is not reported (not applicable for this type of exploratory study). Inclusion/exclusion criteria for cells/traces are not pre-specified (not reported). Outlier handling is not described (not reported). Controls are adequate: untreated cells serve as controls; depletion is validated ('dTAG-13 to acutely deplete the FKBP F36V -tagged endogenous RAD21'), and a second chromosome region (Chr15) is used for validation. Independent replication is not explicitly stated for the key findings as a separate cohort, but replicate experiments are mentioned ('over two replicate experiments for each'). The study uses a large number of cells (300–1700 traces per clone) and 15 cell lines overall, providing some internal replication. However, the lack of pre-registration, blinding, and pre-specified criteria are weaknesses.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionNOT REPORTED

randomization methodNOT REPORTED

replicate distinctionNOT REPORTED

independent replicationINADEQUATE

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 75% conf

Many essential rigor elements for a preclinical interventional study are not reported, including randomization, blinding, power analysis, inclusion/exclusion criteria, outlier handling, and independent replication.

Evidence

direct quote[Results, 'Cohesin accelerates cis-interactions within a TAD']

“To test this hypothesized role for cohesin in these dynamics, we used dTAG-13 to acutely deplete the FKBPF36V-tagged endogenous RAD21, a core component of cohesin, from all 11 of the cell lines and imaged the cohesin-depleted cells in parallel with the untreated controls.”

direct quote[Results, 'High-throughput, ultra-resolution 4D genome imaging']

“Using this approach we imaged all 11 of our initial TRACK-IT clones for 30 minutes each, collecting 300–1700 traces (cells) per clone over two replicate experiments for each.”

absence[Methods Summary]

Randomization, blinding, power analysis, inclusion/exclusion, outlier handling, and independent replication are not reported.

WARNReviewer 2· 85% conf

Key elements such as randomization, blinding, and a pre-specified analysis plan are not reported; while some aspects (controls, inclusion/exclusion) are adequate, the study is a complex observational/imaging study with an interventional component (cohesin depletion) that lacks design transparency.

Evidence

absence[Methods section]

No mention of randomization, blinding, power analysis, or pre-specified inclusion/exclusion criteria.

paraphrase[Results sections 'Cohesin accelerates cis-interactions within a TAD' and 'TAD borders regulate speed and frequency of cis-interactions']

“Untreated cells serve as controls for cohesin depletion; additional experiments on Chr15 validate TAD border effects.”

direct quote[Results section 'High-throughput, ultra-resolution 4D genome imaging']

“We imaged all 11 of our initial TRACK-IT clones for 30 minutes each, collecting 300–1700 traces (cells) per clone over two replicate experiments for each.”

WARNReviewer 3· 85% conf

The study design is observational and involves extensive imaging with multiple controls, but several key design elements such as power analysis, inclusion/exclusion criteria, and outlier handling are not reported.

Evidence

direct quote[Methods, Results]

“To test this hypothesized role for cohesin in these dynamics, we used dTAG-13 to acutely deplete the FKBP F36V -tagged endogenous RAD21, a core component of cohesin, from all 11 of the cell lines and imaged the cohesin-depleted cells in parallel with the untreated controls.”

absence[Methods, Results]

Power analysis is not reported.

absence[Methods, Results]

Inclusion/exclusion criteria for cells or traces are not explicitly reported.

The cell line is described as 'hybrid (CAST/129) mESCs' in the methods, but the sex of the cell line (typically male or female embryonic stem cells) is not reported. Age/health status is not reported for cells (passage number, mycoplasma status not mentioned in the main text). Housing conditions for animals from which cells were derived are not reported (not relevant for ES cell culture). Demographics for human subjects are not applicable. The lack of sex and mycoplasma status are notable gaps for cell culture work.

Sub-criteria

demographicsN/A

sex reportedNOT REPORTED

sex justifiedN/A

age weight healthNOT REPORTED

housing conditionsNOT REPORTED

species strain sourceADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 70% conf

Species/strain and source are adequately reported, but sex of the cell line is not reported, and health status is not explicitly described.

Evidence

direct quote[Results, 'High-throughput, ultra-resolution 4D genome imaging']

“We integrated rTetR-tdStayGold and CymR-2xHaloTag-JF549, a pair of bright, highly photostable DNA binding labels, in hybrid (CAST/129) mESCs”

absence[Results, 'High-throughput, ultra-resolution 4D genome imaging']

Sex of the cell line is not reported.

WARNReviewer 2· 75% conf

The paper reports species and strain (mouse embryonic stem cells, hybrid CAST/129), but does not report sex, age, health status, or housing conditions; demographics are not applicable.

Evidence

direct quote[Methods section - Generation of TRACK-IT plasmids]

“We integrated rTetR-tdStayGold and CymR-2xHaloTag-JF549, a pair of bright, highly photostable DNA binding labels, in hybrid (CAST/129) mESCs”

absence[Methods and Results]

Sex of the ES cell line is not reported.

absence[Methods section]

Age/passage number and health (mycoplasma) status of cell lines are not reported.

PASSReviewer 3· 80% conf

Biological variables (cell type, cell cycle state, chromatin states) are well-characterized, though sex of the mouse embryonic stem cells and housing conditions are not applicable.

Evidence

paraphrase[Methods, Generation and culture of cell lines]

“The paper uses F121-9-CASTx129 mouse embryonic stem cells (mESCs), a hybrid strain.”

direct quote[Results, MSCD measurements obey patterns predicted by polymer physics]

“We imaged asynchronously cycling cells, and distinguished G1 from S/G2 by the presence of doublet foci in replicated S/G2 cells ().”

The paper does not describe any new animal experiments or human subject recruitment. It mentions using 'mouse embryonic stem cells' (mESCs), which are cell lines. No ethics approval is needed for work on established cell lines that are not directly derived from animals in the study. The paper does not claim to have IACUC approval, which is consistent with the lack of animal work. Ethics are genuinely not applicable.

Sub-criteria

iacuc statementN/A

informed consentN/A

irb ethics statementN/A

regulatory complianceN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

The study uses mouse embryonic stem cells, which involve animal research, but no IACUC approval statement is reported; regulatory compliance is also not stated.

Evidence

absence[Methods Summary]

No IACUC approval statement is reported.

absence[Methods Summary]

No regulatory compliance statement is reported.

N/AReviewer 2· 95% conf

The study uses commercially available or previously derived mouse embryonic stem cell lines and does not involve new animal or human subject data acquisition, making ethics approvals not applicable.

Evidence

absence[Entire paper]

No statement of animal or human ethics approval is present, and no new animal or human subject data were collected.

paraphrase[Methods - Generation and culture of cell lines]

“The paper uses hybrid (CAST/129) mESCs and clonal lines generated from them.”

N/AReviewer 3· 95% conf

This study uses established cell lines (not primary human/animal subjects) and reuses publicly available datasets, so no ethics approval is required.

Evidence

paraphrase[Methods and Acknowledgements]

“The study uses mouse embryonic stem cell lines (F121-9-CASTx129) and public GEO datasets, with no mention of new animal or human subject research.”

Among applicable criteria: organisms_identified is adequate (species/strain/source reported). software_tools_identified is reported_but_inadequate (Zenodo links provided but no version numbers). cell_line_authentication is not_reported, reagents_identified is not_reported (no catalog/Addgene IDs for plasmids, transposons, etc.). Mycoplasma testing and antibodies are not applicable. With 1 of 4 applicable criteria adequate (25%), the dimension fails.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedNOT REPORTED

organisms identifiedADEQUATE

antibodies identifiedN/A

cell line authenticationNOT REPORTED

software tools identifiedINADEQUATE

Per reviewer (3) — the votes behind the verdict

FAILReviewer 1· 75% conf

Key resources are inadequately identified: cell line authentication and mycoplasma testing are not reported, reagents lack catalog numbers, and software versions are not specified.

Evidence

direct quote[Data and materials availability]

“Software (data analysis (), microscope control (), and polymer simulations ()) and data () developed for this project are freely available through Zenodo.”

absence[Methods Summary]

Cell line authentication is not reported.

paraphrase[Methods Summary]

“The TRACK-IT construct, Sleeping Beauty transposon, and repressor arrays are named but not given suppliers or catalog numbers.”

WARNReviewer 2· 80% conf

Key resources like plasmids, fluorophores, and software are identified, but antibodies (if any) are not listed, cell line authentication and mycoplasma testing are not reported, and the specific binding proteins (CymR, rTetR) are not fully identified.

Evidence

paraphrase[Data and materials availability section]

“Software (data analysis, microscope control, polymer simulations) and data are available via Zenodo; URLs are provided.”

direct quote[Methods and Acknowledgments]

“We thank Joanna Wysocka for the F121-9-CASTx129 mouse embryonic stem cell parent line.”

paraphrase[Methods and Results]

“dTAG-13 is used for depletion; TetO and CuO arrays are described but no vendor or source catalog numbers are given.”

PASSReviewer 3· 90% conf

Key resources, including software, cell lines, plasmids, and public datasets, are well-identified with accessions and URLs; the investigational product (dTAG-13) is named but without a catalog number.

Evidence

direct quote[Methods, Data and materials availability]

“Software (data analysis (), microscope control (), and polymer simulations ()) and data () developed for this project are freely available through Zenodo..”

direct quote[Results, Cohesin accelerates cis-interactions within a TAD]

“we used dTAG-13 to acutely deplete the FKBP F36V -tagged endogenous RAD21”

direct quote[Data and materials availability]

“Transposon mapping data (isT-seq) were deposited into GEO under accession number GSE289566”

Statistical tests are named: 'Pearson's R =0.98', 'chi-squared test', 'Kaplan-Meier (KM) nonparametric estimator'. Assumptions for the KM estimator are plausible but not explicitly verified (censoring assumed non-informative). Exact p-values are given: 'p =3.6e-3 chi-squared test'. Effect sizes and confidence intervals are reported: 'a= 0.60, 95%-CI=(0.44, 0.76)' for search time scaling. Software is identified: 'Matlab and Python', 'ProcessTraces pipeline', 'polychrom'. Data presentation: mean squared change in distance (MSCD) plots, Kaplan-Meier curves, and individual trace examples are shown; error bars are defined as 95% CI or bootstrapped intervals. Group Ns are reported (e.g., 'n= 21,805 events across the 11 lines'). Mathematical plausibility is not explicitly checkable from aggregate data alone, but no obvious numerical errors are noted in the text. The paper passes the check with adequate reporting.

Sub-criteria

tests namedADEQUATE

exact p valuesADEQUATE

effect sizes ciADEQUATE

data presentationADEQUATE

software identifiedADEQUATE

assumptions verifiedINADEQUATE

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 80% conf

Statistical tests are named, exact p-values are reported in several cases, effect sizes with confidence intervals are provided, and data presentation is appropriate; however, test assumptions are not verified and software versions are not fully specified.

Evidence

direct quote[Results, 'Cohesin extrusion produces processive movement']

“This specificity of processive traces to the untreated population (p=3.6e-3 chi-squared test) further suggests these are cohesin-driven events.”

direct quote[Results, 'Cis-interaction search times suggest motor driven folding']

“the measured search time scaling within the TAD (a= 0.60, 95%-CI=(0.44, 0.76))”

direct quote[Methods Summary]

“All data analysis was performed using Matlab and Python on custom in-house computing clusters.”

PASSReviewer 2· 85% conf

Statistical tests are named (Kaplan-Meier, chi-squared, Pearson's R), exact p-values are reported for key comparisons, effect sizes with confidence intervals are provided for search time scaling, and data presentation is thorough with individual trace data and defined error bars.

Evidence

direct quote[Results section 'Cohesin extrusion produces processive movement']

“This specificity of processive traces to the untreated population ( p =3.6e-3 chi-squared test) further suggests these are cohesin-driven events.”

direct quote[Results section 'Cis-interaction search times suggest motor driven folding']

“The measured search time scaling within the TAD ( a= 0.60, 95%-CI=(0.44, 0.76))”

direct quote[Results section 'High-throughput, ultra-resolution 4D genome imaging']

“The Hi-C and TRACK-IT data correlated quantitatively, (Pearson's R =0.98)”

PASSReviewer 3· 80% conf

Statistical methods are generally well-described, with named tests (Kaplan-Meier, chi-squared, Pearson correlation) and identified software, but effect sizes with confidence intervals are not systematically reported alongside p-values.

Evidence

direct quote[Results, Cohesin extrusion produces processive movement...]

“This specificity of processive traces to the untreated population ( p =3.6e-3 chi-squared test) further suggests these are cohesin-driven events ().”

absence[Results, Cis-interaction search times suggest motor driven folding]

Effect sizes and confidence intervals are not reported for the key search time comparisons.

direct quote[Methods, Image and trajectory Analysis]

“All data analysis was performed using Matlab and Python on custom in-house computing clusters.”

A data availability statement is present. Software and data are deposited on Zenodo with DOIs. Transposon mapping data are in GEO (GSE289566). Code for analysis, microscope control, and polymer simulations is linked via Zenodo. All four sub-criteria are reported_and_adequate.

Sub-criteria

code sharingADEQUATE

accession numbersADEQUATE

repository depositADEQUATE

data availability statementADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

Data and code are made available through public repositories with persistent identifiers: software and data on Zenodo, and transposon mapping data in GEO with accession numbers.

Evidence

direct quote[Data and materials availability]

“Software (data analysis (), microscope control (), and polymer simulations ()) and data () developed for this project are freely available through Zenodo.”

direct quote[Data and materials availability]

“Transposon mapping data (isT-seq) were deposited into GEO under accession number GSE289566”

PASSReviewer 2· 95% conf

The paper provides a comprehensive data and code availability statement with DOIs, a GEO accession number, and links to software repositories; managed access for raw imaging data is not detailed but the statement is concrete.

Evidence

direct quote[Data and materials availability section]

“Software (data analysis (), microscope control (), and polymer simulations ()) and data () developed for this project are freely available through Zenodo.”

direct quote[Data and materials availability section]

“Transposon mapping data (isT-seq) were deposited into GEO under accession number GSE289566”

direct quote[Data and materials availability section]

“Data from prior ChIP-seq analysis for H3K27ac, H3K27me3, H3K36me1, H3K36me3 and CTCF, re-used in this work, is available on GEO GSE146451”

PASSReviewer 3· 95% conf

Data and code are comprehensively shared: software is deposited in Zenodo with URLs, sequencing data are in GEO, and a data availability statement is present and concrete.

Evidence

direct quote[Data and materials availability]

“Software (data analysis (), microscope control (), and polymer simulations ()) and data () developed for this project are freely available through Zenodo..”

direct quote[Data and materials availability]

“Transposon mapping data (isT-seq) were deposited into GEO under accession number GSE289566”

direct quote[Methods, Image and trajectory Analysis]

“Live cell trajectories were generated through our ProcessTraces pipeline written in Matlab and Python, available open source (see ).”

Methods cover cell line generation, imaging, and analysis with pointers to supplements. Trial registration is not applicable. Reporting guidelines (e.g., ARRIVE) are not referenced. All outcomes mentioned (search times, frequencies, contact durations, MSCD) are reported. Limitations are discussed (e.g., assumptions about processive motion, inference of extrusion speed). Conclusions are generally cautious. Funding and competing interests are stated. With 6 of 7 applicable criteria adequate (86%), the dimension passes.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedADEQUATE

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

Methods are described sufficiently with references to supplemental protocols, all key outcomes are reported, limitations are discussed, conclusions are proportional, and funding/COI statements are provided; only a reporting guideline reference is absent.

Evidence

direct quote[Methods Summary]

“For details, see - 1. Optimization of fluorescent labels for DNA tracking.”

direct quote[Discussion, 'The speed of cohesin loop extrusion']

“While we cannot definitely know that processive motion of the DNA is directly driven by a cohesin at the base of the extruded chromatin loop, two lines of evidence support our claim.”

direct quote[Acknowledgments]

“Funding: National institutes of Health grant U01DK127419 (ANB, AS) ... Competing interests: Authors declare that they have no competing interests.”

WARNReviewer 2· 80% conf

Methods are detailed, limitations are discussed, conclusions are proportional, funding and COI are stated, but the paper does not reference a specific reporting guideline (e.g., ARRIVE or a cell biology checklist) and does not register the study in a trial registry (not applicable for this type).

Evidence

direct quote[Funding and Competing interests sections]

“Funding: National institutes of Health grant U01DK127419 (ANB, AS) National Science Foundation grant EF2022182 (AS, ANB) Competing interests: Authors declare that they have no competing interests.”

direct quote[Discussion section]

“While we cannot definitely know that processive motion of the DNA is directly driven by a cohesin at the base of the extruded chromatin loop, two lines of evidence support our claim.”

absence[Methods and Reporting]

No reporting guideline (e.g., ARRIVE) is referenced.

PASSReviewer 3· 90% conf

The manuscript is transparent: methods are detailed, limitations are discussed, conclusions are proportional, and funding/conflicts are stated. Trial registration is not applicable, and a formal reporting guideline is not mentioned.

Evidence

direct quote[Discussion, paragraph 1]

“While we cannot definitely know that processive motion of the DNA is directly driven by a cohesin at the base of the extruded chromatin loop, two lines of evidence support our claim.”

direct quote[Discussion, paragraph 5]

“This suggests that the TADs studied here should be understood as domains of distinctive kinetic s, at least in addition to, if not instead of, being understood as genomic structures , as sometimes depicted () (, ).”

direct quote[Acknowledgments/Funding/Competing interests]

“Competing interests: Authors declare that they have no competing interests.”

13 copyedit issues flagged: mostly clarity, consistency, other.

Reproducibility

1 data/code link checked; 1 live.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

This is a post-publication audit. The published work is robust in its data sharing and foundational reasoning, but the gaps in study design reporting (no blinding, no pre-specified analysis criteria) limit the reader's ability to assess potential biases in the causal claims about cohesin. An informed reader should interpret the quantiative comparisons between conditions with caution; an erratum adding the missing rigor details (blinding status, inclusion/exclusion rules, outlier handling) would strengthen the paper. Independent re-analysis with pre-registered criteria and sample-size justification would increase confidence in the headline conclusions.

1.
HIGHreporting
Add a statement on whether image analysis was blinded to experimental condition (untreated vs. cohesin-depleted); if not feasible, state the rationale.
Blinding reduces the risk of observer bias in trajectory classification and outcome measurement.
2.
HIGHreporting
Define pre-specified inclusion and exclusion criteria for cells and traces (e.g., minimum trace length, signal-to-noise thresholds) in the Methods.
Without explicit criteria, the possibility of post-hoc selection cannot be ruled out, affecting conclusion reliability.
3.
HIGHreporting
Report how outliers (e.g., aberrant trajectories) were handled and whether any data points were excluded with reasons.
Outlier handling directly impacts the statistical results and should be transparent.
4.
HIGHreporting
Clearly distinguish biological replicates (e.g., independent clones or separate experiments) from technical replicates (e.g., multiple cells from the same clone) and state how many of each were used.
The current mention of 'two replicate experiments' is ambiguous and prevents proper assessment of data independence.
5.
HIGHreporting
Conduct and report an a priori power analysis or sample-size justification for the number of cells/traces per condition.
Power analysis supports the adequacy of the sample size to detect the reported effect sizes.
6.
HIGHreporting
Report the sex of the mouse ES cell line and justify if single-sex cells were used.
Sex is a biological variable that can influence chromatin state and gene expression; its omission is a gap for reproducibility.
7.
HIGHreporting
Add a statement on cell line authentication (e.g., STR profiling or validation from source) and mycoplasma testing results for the mESC lines.
Authentication and mycoplasma testing are standard to ensure cell line identity and health, directly affecting data reliability.
8.
HIGHreporting
Provide catalog numbers, RRIDs, or Addgene IDs for all key reagents (dTAG-13, plasmids, transposon constructs, fluorophores).
Full reagent identification enables other laboratories to precisely reproduce the experiments.
9.
MEDIUMreporting
Report software version numbers (e.g., Matlab 2021a, Python 3.9) and ensure the Zenodo deposits include version information.
Version numbers are needed to precisely reproduce computational analyses.
10.
MEDIUMreporting
Reference a reporting guideline (e.g., ARRIVE 2.0 for preclinical animal cell studies) in a dedicated section.
Following a guideline demonstrates adherence to community standards and may improve completeness.
11.
MEDIUMstatistics
Report exact p-values (not thresholds) and effect sizes with 95% confidence intervals for all main comparisons (e.g., search times between conditions, not only the scaling exponent).
Complete reporting of effect estimates and precision allows readers to fully assess the magnitude and uncertainty of findings.
12.
MEDIUMstatistics
Verification: In the results reporting 'p=3.6e-3 chi-squared test', note that expected counts for the chi-squared test may be below 5 (2 vs 20 events in depleted vs untreated); consider confirming with Fisher's exact test.
The low event count may violate chi-squared assumptions; Fisher's exact test is more appropriate and the p-value should be checked.
13.
LOWcopyedit
Fix typo 'kinetic s' to 'kinetics' in Discussion, paragraph 5.
Typos detract from professionalism and clarity.
14.
LOWcopyedit
Standardize hyphenation of 'ultra-resolution' throughout (currently 'ultraresolution' in Abstract).
Consistent terminology improves readability.
15.
LOWcopyedit
Insert proper punctuation in author list and acknowledgments section to separate names.
Current run-together names could cause confusion in attribution.

Full text (44,325 chars)Analyzed: Jul 5, 2026

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