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The single-cell epigenetic regulatory landscape in mammalian perinatal testis development

Jinyue Liao1, Hoi Ching Suen1, Shitao Rao2, Shitao Rao1, Luk Acs1, Zhang R1, Lee Awt1, Chan Tht1, Man Yee Cheung1, Chu Ht1, Hon-Cheong So1, Hobbs Rm3, Tzong-Yi Lee1 •
The Chinese University of Hong Kong1, Fujian Medical University2, Hudson Institute of Medical Research3
17 Mar 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this article, the authors analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis and identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance.
Abstract: Spermatogenesis depends on an orchestrated series of developing events in germ cells and full maturation of the somatic microenvironment. To date, the majority of efforts to study cellular heterogeneity in testis has been focused on single-cell gene expression rather than the chromatin landscape shaping gene expression. To advance our understanding of the regulatory programs underlying testicular cell types, we analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. We showed that scATAC-Seq allowed us to deconvolve distinct cell populations and identify cis-regulatory elements (CREs) underlying cell type specification. We identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed detailed regulatory dynamics coordinating the sequential developmental progressions of germ cells and somatic cells. This high-resolution data also revealed putative stem cells within the Sertoli and Leydig cell populations. Further, we defined candidate target cell types and genes of several GWAS signals, including those associated with testosterone levels and coronary artery disease. Collectively, our data provide a blueprint of the ‘regulon’ of the mouse male germline and supporting somatic cells.

Summary (4 min read)

Jump to: [Introduction] – [Results] – [A. Experimental design. The workflow of testis collection and scATAC-seq to measure] – [D. TF footprints (average ATAC-seq signal around predicted binding sites) for selected] – [B. Heatmaps of differential TF motif activity (left) and gene activity (right) of positive TF] – [TF dynamics during perinatal Sertoli cell development] – [F. Representative confocal images of seminiferous tubules from Oct4-GFP transgenic] – [Discussion] – [Animals] – [Sample collection] – [Cell lysis and tagmentation] – [Sequencing reads preprocessing] – [Trajectory analysis] – [Footprinting analysis] – [Statistical analysis] – [Acknowledgments] and [Author Contributions]

Introduction

  • Mammalian testis consists of germ cells and distinct somatic cell types that coordinately underpin the maintenance of spermatogenesis and fertility.
  • These testicular cells display extensive developmental dynamics during the perinatal period.
  • In mice, Sertoli cells actively proliferate during the neonatal period for two weeks (Vergouwen et al. 1991).
  • Since all cells share the same genetic information, lineage specification must be regulated by differential chromatin accessibility in a cell type-specific and dynamic manner.

Results

  • Single-cell ATAC-Seq captures developmental and cell type-specific heterogeneity in the testis.
  • To delineate the dynamic changes on cellular populations in a developing testis, the authors profiled the chromatin accessibility landscapes of mouse perinatal testis across E18.5 and postnatal stages (P0.5, P2.5 and P5.5) by scATAC-Seq (Figure 1A).
  • These time points were chosen to represent the diversity of cell type compositions involved in the key developmental events in the testis (Figure 1B).
  • Altogether, the authors profiled chromatin accessibility in 25,613 individual cells after stringent quality control filtration and heterotypic doublet removal (Supplementary Figure S1).
  • These samples showed no clustering based on covariates such as transcription start site (TSS) enrichment and fragment size (Supplementary Figure S2A).

A. Experimental design. The workflow of testis collection and scATAC-seq to measure

  • Shared embedding in which cells were grouped by cell type rather than developmental stage (Korsunsky et al. 2019).
  • Prediction of cell types in scATAC-Seq was then performed by directly aligning cells from scATAC-Seq with cells from scRNA-Seq through comparing the ‘query’ gene activity scores matrix with ‘reference’ scRNA-Seq gene expression matrix.
  • Taken together, scATAC-Seq allowed the detection and assignment of cell identities in the developing testis.
  • Chromatin accessibility defines cell types in developing testis Cell types can be distinguished based on whether differentially accessible chromatin regions (DARs) are ‘open’ or ‘closed’.
  • It is The copyright holder for this preprintthis version posted March 17, 2021.

D. TF footprints (average ATAC-seq signal around predicted binding sites) for selected

  • Deconvolution of chromatin accessibility by cell types revealed accessible sites are primarily located in the distal and intron region (>3-kb from TSS), suggesting an enrichment of gene regulatory elements (Supplementary Figure S3A).
  • To test this, the authors used an analytical framework to link distal peaks to genes in cis, based on the coordination of chromatin accessibility and gene expression levels across cells (Figure 3A).
  • The authors analysis also successfully revealed a previously identified functional enhancer as a novel candidate to regulate Sertoli cell marker Wt1 (Figure 3D).
  • Notably, the Dlk1 -Gtl2 locus demonstrated preferential accessibility in stromal and PTM cells.
  • Dmrt1 prevents spermatogonia from undergoing meiosis by repressing Stra8 transcription (Matson et al. 2010).

B. Heatmaps of differential TF motif activity (left) and gene activity (right) of positive TF

  • Trajectory from gonocyte to undifferentiated and then differentiating spermatogonia in P5.5.
  • The second path bypassed the undifferentiated state but passed through the unknown populations and directly reached the differentiating state by P5.5.
  • The authors observed that the motif binding activity of Id4 was initially high but then declined after birth, while that of ETS and Sp/KLF family members increased in spermatogonia (Supplementary Figure S7D).
  • Sohlh2 , which is critical for early spermatogenesis, is more accessible at the late stage (Hao et al. 2008).
  • Cluster 3 peak-to-gene links were more accessible predominantly in T1-ProSG, such as T and Fbxo4 .

TF dynamics during perinatal Sertoli cell development

  • Re-clustering of Sertoli cells revealed 6 cell clusters (Figure 5A).
  • Notably, C1 is largely distinct from other clusters and comprises mainly E18.5, P0.5 and P2.5 cells, suggesting this subpopulation is only present during the embryonic and early neonatal period.
  • The authors then correlated the gene score of a TF to its corresponding TF z-score to reveal TFs enriched at different developmental stages (Figure 5C).
  • In contrast, HIC1 and CEBPD were upregulated at the later stage (Supplementary Figure S10B).
  • Further, induction of C/EBP proteins by cAMP may play a role in FSH-dependent regulation in Sertoli cells (Grønning et al. 1999).

F. Representative confocal images of seminiferous tubules from Oct4-GFP transgenic

  • The authors then identified the positive TF regulators in Sertoli cells.
  • C1 and C3 showed enrichment in different sets of SLC markers.
  • These results suggested there is also large heterogeneity among different TFs in their involvement in different Leydig cell subpopulations.
  • TFs in immune cells Re-clustering of all the immune cells generated 4 cell clusters ((Figure 1D, Supplementary Figure S12A), which can be re-grouped as 3 main groups based on the expression of their corresponding marker genes, including T cells/NK cells (C1 and C2), myeloid cells (C3) and dendritic cells (C4) (Supplementary Figure S12B).
  • The authors then focused on Sertoli cells and CAD traits.

Discussion

  • Understanding the genetic networks that underlie developmental processes requires a comprehensive understanding of the genes involved as well as the regulatory mechanisms that modulate the expression of these genes.
  • More importantly, a significant amount of peak-to-gene links are within known testis enhancer regions, indicating the reliability of their analysis strategy.
  • In contrast, the subsequent rounds of spermatogenesis are derived from Ngn3 -positive undifferentiated spermatogonia (Yoshida et al. 2006), consistent with the high expression of Ngn3 in the undifferentiated spermatogonia cluster in their dataset.
  • A recent study in chicken sex determination suggested a novel stem Sertoli population could be characterized by lower levels of Dmrt1 and Sox9.
  • The authors showed that testicular cell type-specific peaks displayed increased heritability enrichment in cell populations consistent with the known biology and revealed new biological insights.

Animals

  • All the animal experiments were performed according to the protocols approved by the Animal Experiment Ethics Committee (AEEC) of The Chinese University of Hong Kong (CUHK) and followed the Animals (Control of Experiments) Ordinance (Cap. 340) licensed from the Department of Health, the Government of Hong Kong Special Administrative Region.
  • All the mice were housed under a cycle of 12-hour light/dark and kept in ad libitum feeding and controlled the temperature of 22-24°C.
  • Oct4-EGFP transgenic mice and C57BL/6J mice were maintained in CUHK Laboratory Animal Services Centre.

Sample collection

  • The testes were then digested with 1 mg/ml type 4 collagenase , 1 mg/ml hyaluronidase (Sigma-Aldrich) and 5 µg/ml DNase I (Sigma-Aldrich) at 37°C for 20 min with occasional shaking.
  • The suspension was passed through a 40- µm strainer cap (BD Falcon) to yield a uniform single cell suspension.

Cell lysis and tagmentation

  • The lysis buffer was diluted with ATAC-Tween buffer that contains 0.1% Tween-20 as a detergent.
  • Nuclei were resuspended in tagmentation mix, buffered with 1×PBS supplemented with 0.1% BSA and agitated on a ThermoMixer for 30 min at 37 °C.
  • Tagmented nuclei were kept on ice before encapsulation.
  • ScATAC-Seq library preparation and sequencing Tagmented nuclei were loaded onto a ddSEQ Single-Cell Isolator (Bio-Rad).
  • PCR products were purified using Ampure XP beads and quantified on an Agilent Bioanalyzer (G2939BA, Agilent) using the High-Sensitivity DNA kit (5067-4626, Agilent).

Sequencing reads preprocessing

  • Sequencing data were processed using the Bio-Rad ATAC-Seq Analysis Toolkit.
  • The reference index was built upon the mouse genome mm10.
  • For generation of the fragments file, which contain the start and end genomic coordinates of all aligned sequenced fragments, sorted bam files were further process with “bap-frag” module of BAP (https://github.com/caleblareau/bap ).
  • The authors filtered out low-quality nuclei with stringent selection criteria, including read depth per cell (>2,000) and TSS enrichment score (>20%).
  • ArchR was used to estimate gene expression for genes and TF motif activity from single cell chromatin accessibility data.

Trajectory analysis

  • Trajectory analysis was performed in ArchR. addTrajectory function in ArchR was used to construct trajectory on cisTopic UMAP embedding.
  • To perform integrative analyses for identification of positive TF regulators by integration of gene scores with motif accessibility across pseudo-time, the authors used the correlateTrajectories function which takes two SummarizedExperiment objects retrieved from the getTrajectories function.

Footprinting analysis

  • Differential transcription factor footprints across cell types were identified using the Regulatory Genomics Toolbox application HINT (Li et al. 2018).
  • Aligned BAM files from different cell types were treated as pseudo-bulk ATAC-Seq profiles and then subjected to rgt-hint analysis.
  • Based on MACS calling peaks, the authors used HINT-ATAC to predict footprints with the “rgt-hint footprinting” command.
  • The authors then identified all binding sites of a particular TF overlapping with footprints by using its motif from JASPAR with “rgt-motifanalysis matching” command.
  • Differential motif occupancy was identified with “rgt-hint differential” command and ”–bc” was specified to use the bias-corrected signal.

Statistical analysis

  • Assessment of statistical significance was performed using two-tailed unpaired t-tests, one-way ANOVA with Tukey multiple comparisons tests or Chi-squared tests.
  • Statistical analysis was performed using GraphPad Prism v8.

Acknowledgments

  • The authors thank Tommy Lo for scATAC-Seq experimental assistance and Jesse Xiao for interactive website development.
  • The authors also thank the SBS Core Laboratories in The Chinese University of Hong Kong for technical support.

Author Contributions

  • JL, HC Suen, RMH and TLL conceived and designed the study.
  • JL and HC Suen performed experiments, single-cell sequencing analysis and wrote the manuscript.
  • SR, RZ and HC So performed LDSC analysis.
  • JL, HC Suen, RMH and TLL analyzed and interpreted data.
  • JL, HC Suen, ACSL, AWTL, THTC, MYC, HTC, RMH and TLL reviewed and edited the manuscript.

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Figures (5)

Content maybe subject to copyright    Report

The single-cell epigenetic regulatory landscape in mammalian
perinatal testis development
Jinyue LIAO
1,5
, Hoi Ching SUEN
1,5
, Shitao RAO
2,3
, Alfred Chun Shui LUK
1
, Ruoyu ZHANG
2
,
Annie Wing Tung LEE
1
, Ting Hei Thomas CHAN
1
, Man Yee CHEUNG
1
, Ho Ting CHU
1
, Hon
Cheong SO
2
, Robin M. HOBBS
4
*, Tin-Lap LEE
1
*
Author List Footnotes:
1
Developmental and Regenerative Biology Program, School of Biomedical Sciences, Faculty
of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
2
Cancer Biology and Experimental Therapeutics Program, School of Biomedical Sciences,
Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
3
School of Medical Technology and Engineering, Fujian Medical University, Fujian, P.R.
China
4
Germline Stem Cell Biology Laboratory, Centre for Reproductive Health, Hudson Institute of
Medical Research, Australia
5
These authors contributed equally
*Correspondence: Tin-Lap Lee leetl@cuhk.edu.hk; Robin M. Hobbs
robin.hobbs@monash.edu.
1
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.17.435776doi: bioRxiv preprint
Abstract
Spermatogenesis depends on an orchestrated series of developing events in germ cells and
full maturation of the somatic microenvironment. To date, the majority of efforts to study
cellular heterogeneity in testis has been focused on single-cell gene expression rather than
the chromatin landscape shaping gene expression. To advance our understanding of the
regulatory programs underlying testicular cell types, we analyzed single-cell chromatin
accessibility profiles in more than 25,000 cells from mouse developing testis. We showed
that scATAC-Seq allowed us to deconvolve distinct cell populations and identify
cis-regulatory elements (CREs) underlying cell type specification. We identified sets of
transcription factors associated with cell type-specific accessibility, revealing novel
regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed
detailed regulatory dynamics coordinating the sequential developmental progressions of
germ cells and somatic cells. This high-resolution data also revealed putative stem cells
within the Sertoli and Leydig cell populations. Further, we defined candidate target cell types
and genes of several GWAS signals, including those associated with testosterone levels and
coronary artery disease. Collectively, our data provide a blueprint of the ‘regulon’ of the
mouse male germline and supporting somatic cells.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.17.435776doi: bioRxiv preprint
Introduction
Mammalian testis consists of germ cells and distinct somatic cell types that coordinately
underpin the maintenance of spermatogenesis and fertility. These testicular cells display
extensive developmental dynamics during the perinatal period. Primordial germ cells (PGCs)
give rise to M-prospermatogonia at about embryonic day (E) 12, which enter G0 mitotic
arrest at about E14 to form T1-prospermatogonia (T1-ProSG) (McCarrey 2013). Shortly after
birth, T1-ProSG resume mitotic activity and begin migrating from the center of the testis
cords to the basal lamina of testicular cords, and become T2-prospermatogonia (T2-ProSG).
Once resident at the basement membrane, T2-ProSG generate spermatogonia including
self-renewing spermatogonial stem cells (SSCs) or directly transition into differentiating
spermatogonia that participate in the first round of spermatogenesis (Kluin and de Rooij
1981; Manku and Culty 2015).
Specialized somatic cells play a pivotal role in maintaining normal germ cell development
and spermatogenesis. SSCs and their initial progenies reside in a niche on the basement
membrane and are surrounded by Sertoli cells, which nourish SSCs. In mice, Sertoli cells
actively proliferate during the neonatal period for two weeks (Vergouwen et al. 1991).
Outside the seminiferous tubules, the interstitial compartment of testis mainly contains
stroma, peritubular myoid cells (PTMs), Leydig cells, macrophages and vascular cells.
PTMs are smooth muscle cells that distribute over the peripheral surface of the basement
membrane (Maekawa et al. 1996). They are mainly involved in tubule contractions to
facilitate the movement of sperm to the epididymis and secrete extracellular matrix materials
(Chen et al. 2014). Leydig cells are responsible for steroidogenesis and provide critical
support for spermatogenesis. In mammals, there are two types of Leydig cells, fetal Leydig
cells (FLCs) and adult Leydig cells (ALCs), which develop sequentially in the testis (Shima
2019). During the perinatal period, both FLCs and stem Leydig cells (SLCs) reside in the
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.17.435776doi: bioRxiv preprint
testis. While FLCs start to degenerate after birth, they are replaced by SLCs, which are the
progenitors of ALCs (Su et al. 2018).
One of the goals of developmental biology is to identify transcriptional networks that regulate
cell differentiation. Recent advances in single-cell transcriptomic methods have enabled an
unbiased identification of cell types and gene expression networks in testis (Green et al.
2018; Tan et al. 2020). A key remaining question is how distinct testis cell types are
developed during the perinatal period. Since all cells share the same genetic information,
lineage specification must be regulated by differential chromatin accessibility in a cell
type-specific and dynamic manner. Thus, the understanding of the testis development,
especially the cell type-specific transcription factors (TFs) is of paramount importance.
scRNA-Seq provides limited information of TFs, which are usually lowly expressed. Although
sequencing methods such as ATAC-Seq and DNase-Seq have been developed for profiling
chromatin accessibility landscapes across samples and classification of regulatory elements
in the genome, the nature of bulk measurements masks the cellular and regulatory
heterogeneity in subpopulations within a given cell type (Shema et al. 2019). Moreover,
uncovering regulatory elements in testicular cell types has been particularly challenging as
samples are limited and heterogeneous. Notably, only one previous study examined the
genome-wide chromatin accessibility in Sertoli cells, using the Sox9 transgenic line followed
by fluorescence-activated cell sorting (FACS) and DNase-Seq (Maatouk et al. 2017).
By profiling the genome-wide regulatory landscapes at a single-cell level, recent single-cell
sequencing assay for transposase-accessible chromatin (scATAC-Seq) studies have
demonstrated the potential to discover complex cell populations, link regulatory elements to
their target genes, and map regulatory dynamics during complex cellular differentiation
processes. We reasoned that the advent of new single-cell chromatin accessibility
sequencing methods, combined with single-cell transcriptomic data, would be instrumental in
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.17.435776doi: bioRxiv preprint
advancing our understanding of gene regulatory networks in mammalian testis development.
Here, we applied scATAC-Seq to deconvolve cell populations and identify cell type-specific
epigenetic regulatory circuits during perinatal testis development. The dataset led to
identification of key cell type-specific TFs, defined the cellular differentiation trajectory, and
characterized regulatory dynamics of distinct cell types. Furthermore, our results shed light
on the identification of target cell types for genetic variants. To enable public access to our
data, we constructed the mouse testis epigenetic regulatory atlas website at
http://testisatlas.s3-website-us-west-2.amazonaws.com/.
Results
Single-cell ATAC-Seq captures developmental and cell
type-specific heterogeneity in the testis
To delineate the dynamic changes on cellular populations in a developing testis, we profiled
the chromatin accessibility landscapes of mouse perinatal testis across E18.5 and postnatal
stages (P0.5, P2.5 and P5.5) by scATAC-Seq (Figure 1A). These time points were chosen to
represent the diversity of cell type compositions involved in the key developmental events in
the testis (Figure 1B). Altogether, we profiled chromatin accessibility in 25,613 individual cells
after stringent quality control filtration and heterotypic doublet removal (Supplementary
Figure S1). These samples showed no clustering based on covariates such as transcription
start site (TSS) enrichment and fragment size (Supplementary Figure S2A).
Several clusters showed developmental stage specificity, which were made up almost
entirely of cells from a single time point (Supplementary Figure S2B). To improve cell type
annotation, we used Harmony to integrate datasets of 4 time points and project cells onto a
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 17, 2021. ; https://doi.org/10.1101/2021.03.17.435776doi: bioRxiv preprint
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Dynamic Gene Regulatory Networks of Human Myeloid Differentiation.

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26 Apr 2017-Cell systems
Ricardo Ramirez, Nicole C. El-Ali, Mikayla Anne Mager, Dana Wyman, Ana Conesa, Ali Mortazavi 
Single-Cell Analysis of Human Retina Identifies Evolutionarily Conserved and Species-Specific Mechanisms Controlling Development

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18 May 2020-Developmental Cell
Yufeng Lu, Fion Shiau, Wenyang Yi, Suying Lu, Qian Wu, Joel Pearson, Alyssa Kallman, Suijuan Zhong, Thanh Hoang, Zhentao Zuo, Fangqi Zhao, Mei Zhang, Mei Zhang, Nicole Tsai, Yan Zhuo, Sheng He, Jun Zhang, Genevieve Stein-O’Brien, Thomas D. Sherman, Xin Duan, Elana J. Fertig, Loyal A. Goff, Loyal A. Goff, Donald J. Zack, James T. Handa, Tian Xue, Tian Xue, Rod Bremner, Seth Blackshaw, Xiaoqun Wang, Brian S. Clark 
Identifying transposable element expression dynamics and heterogeneity during development at the single-cell level with a processing pipeline scTE.

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05 Mar 2021-Nature Communications
Jiangping He, Isaac A. Babarinde, Li Sun, Shuyang Xu, Ruhai Chen, Junjie Shi, Yuanjie Wei, Yuhao Li, Gang Ma, Qiang Zhuang, Andrew P. Hutchins, Jiekai Chen 
Frequently Asked Questions (15)
Q1. What are the contributions mentioned in the paper "The single-cell epigenetic regulatory landscape in mammalian perinatal testis development" ?

To advance their understanding of the regulatory programs underlying testicular cell types, the authors analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. The authors showed that scATAC-Seq allowed us to deconvolve distinct cell populations and identify cis-regulatory elements ( CREs ) underlying cell type specification. 4. 0 International license made available under a ( which was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Further, the authors defined candidate target cell types and genes of several GWAS signals, including those associated with testosterone levels and coronary artery disease. 

Assessment of statistical significance was performed using two-tailed unpaired t-tests, one-way ANOVA with Tukey multiple comparisons tests or Chi-squared tests. 

As enhancers play a critical role in establishing tissue-specific gene expression patterns during development, the authors predicted that active enhancers would be enriched around lineage-specific genes. 

Shortly after birth, T1-ProSG resume mitotic activity and begin migrating from the center of the testis cords to the basal lamina of testicular cords, and become T2-prospermatogonia (T2-ProSG). 

Bin regions were cleaned by eliminating bins overlapping with ENCODE Blacklist regions, mitochondrial DNA as well as the top 5% of invariant features (house-keeping gene promoters). 

They are mainly involved in tubule contractions to facilitate the movement of sperm to the epididymis and secrete extracellular matrix materials (Chen et al. 2014). 

For generation of the fragments file, which contain the start and end genomic coordinates of all aligned sequenced fragments, sorted bam files were further process with “bap-frag” module of BAP (https://github.com/caleblareau/bap ). 

Since there is currently a lack of marker genes to accurately identify gonocytes which undergo the first wave of spermatogenesis, their scATAC-Seq data uncovered a list of potential markers that warrants further investigation . 

GO terms associated with the marker genes in C1 include extracellular structure organization and connective tissue development (Supplementary Figure S10G). 

Differential motif occupancy was identified with “rgt-hint differential” command and ”–bc” was specified to use the bias-corrected signal. 

761 marker genes were identified across the clusters (FDR < 0.1, log2FC > 0.5), with most upregulated in C1 (Supplementary Figure S10F). 

Chromatin accessibility defines cell types in developing testisCell types can be distinguished based on whether differentially accessible chromatin regions (DARs) are ‘open’ or ‘closed’. 

the authors showed that similar to scRNA-Seq, the chromatin accessibility information obtained from scATAC-Seq is able to define cell types in the testis. 

Their scATAC-Seq provides additional information on TF regulation by correlating motif accessibility and predicted gene activity, which allows us to reveal cell type-specific TFs. 

Based on the inferred gene scores calculated by ArchR, 81 marker genes were identified among the clusters (FDR < 0.1, log 2FC > 0.5) (Supplementary Figure S10A).