SRX3545454 GSM2917200 SRP128582 SRS2818902 GSM2917200 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917200 GSM2917200 GEO Accession GSM2917200 SRX3545455 GSM2917201 SRP128582 SRS2818901 GSM2917201 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917201 GSM2917201 GEO Accession GSM2917201 SRX3545456 GSM2917202 SRP128582 SRS2818903 GSM2917202 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917202 GSM2917202 GEO Accession GSM2917202 SRX3545457 GSM2917203 SRP128582 SRS2818904 GSM2917203 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917203 GSM2917203 GEO Accession GSM2917203 SRX3545458 GSM2917204 SRP128582 SRS2818905 GSM2917204 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917204 GSM2917204 GEO Accession GSM2917204 SRX3545459 GSM2917205 SRP128582 SRS2818985 GSM2917205 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917205 GSM2917205 GEO Accession GSM2917205 SRX3545460 GSM2917206 SRP128582 SRS2818987 GSM2917206 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917206 GSM2917206 GEO Accession GSM2917206 SRX3545461 GSM2917207 SRP128582 SRS2818986 GSM2917207 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917207 GSM2917207 GEO Accession GSM2917207 SRX3545462 GSM2917208 SRP128582 SRS2818988 GSM2917208 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917208 GSM2917208 GEO Accession GSM2917208 SRX3545463 GSM2917209 SRP128582 SRS2818989 GSM2917209 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917209 GSM2917209 GEO Accession GSM2917209 SRX3545464 GSM2917210 SRP128582 SRS2818990 GSM2917210 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917210 GSM2917210 GEO Accession GSM2917210 SRX3545465 GSM2917211 SRP128582 SRS2818992 GSM2917211 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917211 GSM2917211 GEO Accession GSM2917211 SRX3545466 GSM2917212 SRP128582 SRS2818991 GSM2917212 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917212 GSM2917212 GEO Accession GSM2917212 SRX3545467 GSM2917213 SRP128582 SRS2818993 GSM2917213 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917213 GSM2917213 GEO Accession GSM2917213 SRX3545468 GSM2917214 SRP128582 SRS2818995 GSM2917214 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917214 GSM2917214 GEO Accession GSM2917214 SRX3545469 GSM2917215 SRP128582 SRS2818994 GSM2917215 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917215 GSM2917215 GEO Accession GSM2917215 SRX3545470 GSM2917216 SRP128582 SRS2818996 GSM2917216 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917216 GSM2917216 GEO Accession GSM2917216 SRX3545471 GSM2917217 SRP128582 SRS2818997 GSM2917217 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917217 GSM2917217 GEO Accession GSM2917217 SRX3545472 GSM2917218 SRP128582 SRS2818998 GSM2917218 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917218 GSM2917218 GEO Accession GSM2917218 SRX3545473 GSM2917219 SRP128582 SRS2818999 GSM2917219 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917219 GSM2917219 GEO Accession GSM2917219 SRX3545474 GSM2917220 SRP128582 SRS2819000 GSM2917220 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917220 GSM2917220 GEO Accession GSM2917220 SRX3545475 GSM2917221 SRP128582 SRS2819001 GSM2917221 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917221 GSM2917221 GEO Accession GSM2917221 SRX3545476 GSM2917222 SRP128582 SRS2819003 GSM2917222 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917222 GSM2917222 GEO Accession GSM2917222 SRX3545477 GSM2917223 SRP128582 SRS2819002 GSM2917223 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917223 GSM2917223 GEO Accession GSM2917223 SRX3545478 GSM2917224 SRP128582 SRS2819004 GSM2917224 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917224 GSM2917224 GEO Accession GSM2917224 SRX3545479 GSM2917225 SRP128582 SRS2819005 GSM2917225 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917225 GSM2917225 GEO Accession GSM2917225 SRX3545480 GSM2917226 SRP128582 SRS2819006 GSM2917226 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917226 GSM2917226 GEO Accession GSM2917226 SRX3545481 GSM2917227 SRP128582 SRS2819007 GSM2917227 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917227 GSM2917227 GEO Accession GSM2917227 SRX3545482 GSM2917228 SRP128582 SRS2819009 GSM2917228 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917228 GSM2917228 GEO Accession GSM2917228 SRX3545483 GSM2917229 SRP128582 SRS2819008 GSM2917229 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917229 GSM2917229 GEO Accession GSM2917229 SRX3545484 GSM2917230 SRP128582 SRS2819010 GSM2917230 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917230 GSM2917230 GEO Accession GSM2917230 SRX3545485 GSM2917231 SRP128582 SRS2819012 GSM2917231 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917231 GSM2917231 GEO Accession GSM2917231 SRX3545486 GSM2917232 SRP128582 SRS2819013 GSM2917232 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917232 GSM2917232 GEO Accession GSM2917232 SRX3545487 GSM2917233 SRP128582 SRS2819011 GSM2917233 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917233 GSM2917233 GEO Accession GSM2917233 SRX3545488 GSM2917234 SRP128582 SRS2819016 GSM2917234 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917234 GSM2917234 GEO Accession GSM2917234 SRX3545489 GSM2917235 SRP128582 SRS2819014 GSM2917235 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917235 GSM2917235 GEO Accession GSM2917235 SRX3545490 GSM2917236 SRP128582 SRS2819015 GSM2917236 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917236 GSM2917236 GEO Accession GSM2917236 SRX3545491 GSM2917237 SRP128582 SRS2819017 GSM2917237 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917237 GSM2917237 GEO Accession GSM2917237 SRX3545492 GSM2917238 SRP128582 SRS2819019 GSM2917238 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917238 GSM2917238 GEO Accession GSM2917238 SRX3545493 GSM2917239 SRP128582 SRS2819018 GSM2917239 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917239 GSM2917239 GEO Accession GSM2917239 SRX3545494 GSM2917240 SRP128582 SRS2819020 GSM2917240 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917240 GSM2917240 GEO Accession GSM2917240 SRX3545495 GSM2917241 SRP128582 SRS2819022 GSM2917241 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917241 GSM2917241 GEO Accession GSM2917241 SRX3545496 GSM2917242 SRP128582 SRS2819021 GSM2917242 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917242 GSM2917242 GEO Accession GSM2917242 SRX3545497 GSM2917243 SRP128582 SRS2819023 GSM2917243 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917243 GSM2917243 GEO Accession GSM2917243 SRX3545498 GSM2917244 SRP128582 SRS2819024 GSM2917244 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917244 GSM2917244 GEO Accession GSM2917244 SRX3545499 GSM2917245 SRP128582 SRS2819025 GSM2917245 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917245 GSM2917245 GEO Accession GSM2917245 SRX3545500 GSM2917246 SRP128582 SRS2819026 GSM2917246 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917246 GSM2917246 GEO Accession GSM2917246 SRX3545501 GSM2917247 SRP128582 SRS2819027 GSM2917247 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917247 GSM2917247 GEO Accession GSM2917247 SRX3545502 GSM2917248 SRP128582 SRS2819028 GSM2917248 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917248 GSM2917248 GEO Accession GSM2917248 SRX3545503 GSM2917249 SRP128582 SRS2819029 GSM2917249 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917249 GSM2917249 GEO Accession GSM2917249 SRX3545504 GSM2917250 SRP128582 SRS2819030 GSM2917250 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917250 GSM2917250 GEO Accession GSM2917250 SRX3545505 GSM2917251 SRP128582 SRS2819031 GSM2917251 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917251 GSM2917251 GEO Accession GSM2917251 SRX3545506 GSM2917252 SRP128582 SRS2819032 GSM2917252 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917252 GSM2917252 GEO Accession GSM2917252 SRX3545507 GSM2917253 SRP128582 SRS2819033 GSM2917253 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917253 GSM2917253 GEO Accession GSM2917253 SRX3545508 GSM2917254 SRP128582 SRS2819034 GSM2917254 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917254 GSM2917254 GEO Accession GSM2917254 SRX3545509 GSM2917255 SRP128582 SRS2819035 GSM2917255 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917255 GSM2917255 GEO Accession GSM2917255 SRX3545510 GSM2917256 SRP128582 SRS2819036 GSM2917256 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917256 GSM2917256 GEO Accession GSM2917256 SRX3545511 GSM2917257 SRP128582 SRS2819037 GSM2917257 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917257 GSM2917257 GEO Accession GSM2917257 SRX3545512 GSM2917258 SRP128582 SRS2819038 GSM2917258 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917258 GSM2917258 GEO Accession GSM2917258 SRX3545513 GSM2917259 SRP128582 SRS2819039 GSM2917259 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917259 GSM2917259 GEO Accession GSM2917259 SRX3545514 GSM2917260 SRP128582 SRS2819041 GSM2917260 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917260 GSM2917260 GEO Accession GSM2917260 SRX3545515 GSM2917261 SRP128582 SRS2819040 GSM2917261 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917261 GSM2917261 GEO Accession GSM2917261 SRX3545516 GSM2917262 SRP128582 SRS2819042 GSM2917262 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917262 GSM2917262 GEO Accession GSM2917262 SRX3545517 GSM2917263 SRP128582 SRS2819043 GSM2917263 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917263 GSM2917263 GEO Accession GSM2917263 SRX3545518 GSM2917264 SRP128582 SRS2819044 GSM2917264 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917264 GSM2917264 GEO Accession GSM2917264 SRX3545519 GSM2917265 SRP128582 SRS2819045 GSM2917265 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917265 GSM2917265 GEO Accession GSM2917265 SRX3545520 GSM2917266 SRP128582 SRS2819046 GSM2917266 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917266 GSM2917266 GEO Accession GSM2917266 SRX3545521 GSM2917267 SRP128582 SRS2819047 GSM2917267 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917267 GSM2917267 GEO Accession GSM2917267 SRX3545522 GSM2917268 SRP128582 SRS2819048 GSM2917268 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917268 GSM2917268 GEO Accession GSM2917268 SRX3545523 GSM2917269 SRP128582 SRS2819049 GSM2917269 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917269 GSM2917269 GEO Accession GSM2917269 SRX3545524 GSM2917270 SRP128582 SRS2819050 GSM2917270 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917270 GSM2917270 GEO Accession GSM2917270 SRX3545525 GSM2917271 SRP128582 SRS2819051 GSM2917271 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917271 GSM2917271 GEO Accession GSM2917271 SRX3545526 GSM2917272 SRP128582 SRS2819054 GSM2917272 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917272 GSM2917272 GEO Accession GSM2917272 SRX3545527 GSM2917273 SRP128582 SRS2819053 GSM2917273 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917273 GSM2917273 GEO Accession GSM2917273 SRX3545528 GSM2917274 SRP128582 SRS2819052 GSM2917274 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917274 GSM2917274 GEO Accession GSM2917274 SRX3545529 GSM2917275 SRP128582 SRS2819056 GSM2917275 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917275 GSM2917275 GEO Accession GSM2917275 SRX3545530 GSM2917276 SRP128582 SRS2819057 GSM2917276 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917276 GSM2917276 GEO Accession GSM2917276 SRX3545531 GSM2917277 SRP128582 SRS2819055 GSM2917277 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917277 GSM2917277 GEO Accession GSM2917277 SRX3545532 GSM2917278 SRP128582 SRS2819058 GSM2917278 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917278 GSM2917278 GEO Accession GSM2917278 SRX3545533 GSM2917279 SRP128582 SRS2819059 GSM2917279 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917279 GSM2917279 GEO Accession GSM2917279 SRX3545534 GSM2917280 SRP128582 SRS2819060 GSM2917280 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917280 GSM2917280 GEO Accession GSM2917280 SRX3545535 GSM2917281 SRP128582 SRS2819061 GSM2917281 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917281 GSM2917281 GEO Accession GSM2917281 SRX3545536 GSM2917282 SRP128582 SRS2819062 GSM2917282 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917282 GSM2917282 GEO Accession GSM2917282 SRX3545537 GSM2917283 SRP128582 SRS2819063 GSM2917283 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917283 GSM2917283 GEO Accession GSM2917283 SRX3545538 GSM2917284 SRP128582 SRS2819064 GSM2917284 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917284 GSM2917284 GEO Accession GSM2917284 SRX3545539 GSM2917285 SRP128582 SRS2819065 GSM2917285 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917285 GSM2917285 GEO Accession GSM2917285 SRX3545540 GSM2917286 SRP128582 SRS2819066 GSM2917286 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917286 GSM2917286 GEO Accession GSM2917286 SRX3545541 GSM2917287 SRP128582 SRS2819067 GSM2917287 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917287 GSM2917287 GEO Accession GSM2917287 SRX3545542 GSM2917288 SRP128582 SRS2819069 GSM2917288 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917288 GSM2917288 GEO Accession GSM2917288 SRX3545543 GSM2917289 SRP128582 SRS2819068 GSM2917289 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917289 GSM2917289 GEO Accession GSM2917289 SRX3545544 GSM2917290 SRP128582 SRS2819070 GSM2917290 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917290 GSM2917290 GEO Accession GSM2917290 SRX3545545 GSM2917291 SRP128582 SRS2819071 GSM2917291 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917291 GSM2917291 GEO Accession GSM2917291 SRX3545546 GSM2917292 SRP128582 SRS2819072 GSM2917292 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917292 GSM2917292 GEO Accession GSM2917292 SRX3545547 GSM2917293 SRP128582 SRS2819073 GSM2917293 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917293 GSM2917293 GEO Accession GSM2917293 SRX3545548 GSM2917294 SRP128582 SRS2819074 GSM2917294 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917294 GSM2917294 GEO Accession GSM2917294 SRX3545549 GSM2917295 SRP128582 SRS2819075 GSM2917295 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917295 GSM2917295 GEO Accession GSM2917295 SRX3545550 GSM2917296 SRP128582 SRS2819076 GSM2917296 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917296 GSM2917296 GEO Accession GSM2917296 SRX3545551 GSM2917297 SRP128582 SRS2819077 GSM2917297 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917297 GSM2917297 GEO Accession GSM2917297 SRX3545552 GSM2917298 SRP128582 SRS2819153 GSM2917298 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917298 GSM2917298 GEO Accession GSM2917298 SRX3545553 GSM2917299 SRP128582 SRS2819078 GSM2917299 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917299 GSM2917299 GEO Accession GSM2917299 SRX3545554 GSM2917300 SRP128582 SRS2819079 GSM2917300 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917300 GSM2917300 GEO Accession GSM2917300 SRX3545555 GSM2917301 SRP128582 SRS2819080 GSM2917301 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917301 GSM2917301 GEO Accession GSM2917301 SRX3545556 GSM2917302 SRP128582 SRS2819081 GSM2917302 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917302 GSM2917302 GEO Accession GSM2917302 SRX3545557 GSM2917303 SRP128582 SRS2819082 GSM2917303 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917303 GSM2917303 GEO Accession GSM2917303 SRX3545558 GSM2917304 SRP128582 SRS2819083 GSM2917304 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917304 GSM2917304 GEO Accession GSM2917304 SRX3545559 GSM2917305 SRP128582 SRS2819085 GSM2917305 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917305 GSM2917305 GEO Accession GSM2917305 SRX3545560 GSM2917306 SRP128582 SRS2819084 GSM2917306 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917306 GSM2917306 GEO Accession GSM2917306 SRX3545561 GSM2917307 SRP128582 SRS2819086 GSM2917307 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917307 GSM2917307 GEO Accession GSM2917307 SRX3545562 GSM2917308 SRP128582 SRS2819087 GSM2917308 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917308 GSM2917308 GEO Accession GSM2917308 SRX3545563 GSM2917309 SRP128582 SRS2819088 GSM2917309 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917309 GSM2917309 GEO Accession GSM2917309 SRX3545564 GSM2917310 SRP128582 SRS2819089 GSM2917310 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917310 GSM2917310 GEO Accession GSM2917310 SRX3545565 GSM2917311 SRP128582 SRS2819090 GSM2917311 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917311 GSM2917311 GEO Accession GSM2917311 SRX3545566 GSM2917312 SRP128582 SRS2819091 GSM2917312 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917312 GSM2917312 GEO Accession GSM2917312 SRX3545567 GSM2917313 SRP128582 SRS2819092 GSM2917313 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917313 GSM2917313 GEO Accession GSM2917313 SRX3545568 GSM2917314 SRP128582 SRS2819093 GSM2917314 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917314 GSM2917314 GEO Accession GSM2917314 SRX3545569 GSM2917315 SRP128582 SRS2819094 GSM2917315 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917315 GSM2917315 GEO Accession GSM2917315 SRX3545570 GSM2917316 SRP128582 SRS2819095 GSM2917316 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917316 GSM2917316 GEO Accession GSM2917316 SRX3545571 GSM2917317 SRP128582 SRS2819096 GSM2917317 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917317 GSM2917317 GEO Accession GSM2917317 SRX3545572 GSM2917318 SRP128582 SRS2819097 GSM2917318 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917318 GSM2917318 GEO Accession GSM2917318 SRX3545573 GSM2917319 SRP128582 SRS2819098 GSM2917319 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917319 GSM2917319 GEO Accession GSM2917319 SRX3545574 GSM2917320 SRP128582 SRS2819099 GSM2917320 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917320 GSM2917320 GEO Accession GSM2917320 SRX3545575 GSM2917321 SRP128582 SRS2819100 GSM2917321 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917321 GSM2917321 GEO Accession GSM2917321 SRX3545576 GSM2917322 SRP128582 SRS2819101 GSM2917322 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917322 GSM2917322 GEO Accession GSM2917322 SRX3545577 GSM2917323 SRP128582 SRS2819102 GSM2917323 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917323 GSM2917323 GEO Accession GSM2917323 SRX3545578 GSM2917324 SRP128582 SRS2819103 GSM2917324 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917324 GSM2917324 GEO Accession GSM2917324 SRX3545579 GSM2917325 SRP128582 SRS2819104 GSM2917325 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917325 GSM2917325 GEO Accession GSM2917325 SRX3545580 GSM2917326 SRP128582 SRS2819105 GSM2917326 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917326 GSM2917326 GEO Accession GSM2917326 SRX3545581 GSM2917327 SRP128582 SRS2819107 GSM2917327 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917327 GSM2917327 GEO Accession GSM2917327 SRX3545582 GSM2917328 SRP128582 SRS2819106 GSM2917328 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917328 GSM2917328 GEO Accession GSM2917328 SRX3545583 GSM2917329 SRP128582 SRS2819108 GSM2917329 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917329 GSM2917329 GEO Accession GSM2917329 SRX3545584 GSM2917330 SRP128582 SRS2819109 GSM2917330 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917330 GSM2917330 GEO Accession GSM2917330 SRX3545585 GSM2917331 SRP128582 SRS2819113 GSM2917331 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917331 GSM2917331 GEO Accession GSM2917331 SRX3545586 GSM2917332 SRP128582 SRS2819110 GSM2917332 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917332 GSM2917332 GEO Accession GSM2917332 SRX3545587 GSM2917333 SRP128582 SRS2819111 GSM2917333 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917333 GSM2917333 GEO Accession GSM2917333 SRX3545588 GSM2917334 SRP128582 SRS2819112 GSM2917334 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917334 GSM2917334 GEO Accession GSM2917334 SRX3545589 GSM2917335 SRP128582 SRS2819115 GSM2917335 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917335 GSM2917335 GEO Accession GSM2917335 SRX3545590 GSM2917336 SRP128582 SRS2819116 GSM2917336 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917336 GSM2917336 GEO Accession GSM2917336 SRX3545591 GSM2917337 SRP128582 SRS2819114 GSM2917337 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917337 GSM2917337 GEO Accession GSM2917337 SRX3545592 GSM2917338 SRP128582 SRS2819117 GSM2917338 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917338 GSM2917338 GEO Accession GSM2917338 SRX3545593 GSM2917339 SRP128582 SRS2819118 GSM2917339 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917339 GSM2917339 GEO Accession GSM2917339 SRX3545594 GSM2917340 SRP128582 SRS2819120 GSM2917340 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917340 GSM2917340 GEO Accession GSM2917340 SRX3545595 GSM2917341 SRP128582 SRS2819121 GSM2917341 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917341 GSM2917341 GEO Accession GSM2917341 SRX3545596 GSM2917342 SRP128582 SRS2819119 GSM2917342 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917342 GSM2917342 GEO Accession GSM2917342 SRX3545597 GSM2917343 SRP128582 SRS2819122 GSM2917343 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917343 GSM2917343 GEO Accession GSM2917343 SRX3545598 GSM2917344 SRP128582 SRS2819123 GSM2917344 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917344 GSM2917344 GEO Accession GSM2917344 SRX3545599 GSM2917345 SRP128582 SRS2819124 GSM2917345 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917345 GSM2917345 GEO Accession GSM2917345 SRX3545600 GSM2917346 SRP128582 SRS2819125 GSM2917346 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917346 GSM2917346 GEO Accession GSM2917346 SRX3545601 GSM2917347 SRP128582 SRS2819126 GSM2917347 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917347 GSM2917347 GEO Accession GSM2917347 SRX3545602 GSM2917348 SRP128582 SRS2819127 GSM2917348 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917348 GSM2917348 GEO Accession GSM2917348 SRX3545603 GSM2917349 SRP128582 SRS2819129 GSM2917349 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917349 GSM2917349 GEO Accession GSM2917349 SRX3545604 GSM2917350 SRP128582 SRS2819128 GSM2917350 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917350 GSM2917350 GEO Accession GSM2917350 SRX3545605 GSM2917351 SRP128582 SRS2819130 GSM2917351 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917351 GSM2917351 GEO Accession GSM2917351 SRX3545606 GSM2917352 SRP128582 SRS2819131 GSM2917352 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917352 GSM2917352 GEO Accession GSM2917352 SRX3545607 GSM2917353 SRP128582 SRS2819132 GSM2917353 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917353 GSM2917353 GEO Accession GSM2917353 SRX3545608 GSM2917354 SRP128582 SRS2819133 GSM2917354 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917354 GSM2917354 GEO Accession GSM2917354 SRX3545609 GSM2917355 SRP128582 SRS2819135 GSM2917355 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917355 GSM2917355 GEO Accession GSM2917355 SRX3545610 GSM2917356 SRP128582 SRS2819134 GSM2917356 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917356 GSM2917356 GEO Accession GSM2917356 SRX3545611 GSM2917357 SRP128582 SRS2819136 GSM2917357 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917357 GSM2917357 GEO Accession GSM2917357 SRX3545612 GSM2917358 SRP128582 SRS2819137 GSM2917358 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917358 GSM2917358 GEO Accession GSM2917358 SRX3545613 GSM2917359 SRP128582 SRS2819138 GSM2917359 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917359 GSM2917359 GEO Accession GSM2917359 SRX3545614 GSM2917360 SRP128582 SRS2819139 GSM2917360 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917360 GSM2917360 GEO Accession GSM2917360 SRX3545615 GSM2917361 SRP128582 SRS2819140 GSM2917361 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917361 GSM2917361 GEO Accession GSM2917361 SRX3545616 GSM2917362 SRP128582 SRS2819142 GSM2917362 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917362 GSM2917362 GEO Accession GSM2917362 SRX3545617 GSM2917363 SRP128582 SRS2819141 GSM2917363 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917363 GSM2917363 GEO Accession GSM2917363 SRX3545618 GSM2917364 SRP128582 SRS2819143 GSM2917364 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917364 GSM2917364 GEO Accession GSM2917364 SRX3545619 GSM2917365 SRP128582 SRS2819144 GSM2917365 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917365 GSM2917365 GEO Accession GSM2917365 SRX3545620 GSM2917366 SRP128582 SRS2819145 GSM2917366 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917366 GSM2917366 GEO Accession GSM2917366 SRX3545621 GSM2917367 SRP128582 SRS2819147 GSM2917367 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917367 GSM2917367 GEO Accession GSM2917367 SRX3545622 GSM2917368 SRP128582 SRS2819146 GSM2917368 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917368 GSM2917368 GEO Accession GSM2917368 SRX3545623 GSM2917369 SRP128582 SRS2819148 GSM2917369 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917369 GSM2917369 GEO Accession GSM2917369 SRX3545624 GSM2917370 SRP128582 SRS2819149 GSM2917370 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917370 GSM2917370 GEO Accession GSM2917370 SRX3545625 GSM2917371 SRP128582 SRS2819150 GSM2917371 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917371 GSM2917371 GEO Accession GSM2917371 SRX3545626 GSM2917372 SRP128582 SRS2819151 GSM2917372 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917372 GSM2917372 GEO Accession GSM2917372 SRX3545627 GSM2917373 SRP128582 SRS2819152 GSM2917373 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917373 GSM2917373 GEO Accession GSM2917373 SRX3545628 GSM2917374 SRP128582 SRS2819154 GSM2917374 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917374 GSM2917374 GEO Accession GSM2917374 SRX3545629 GSM2917375 SRP128582 SRS2819155 GSM2917375 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917375 GSM2917375 GEO Accession GSM2917375 SRX3545630 GSM2917376 SRP128582 SRS2819156 GSM2917376 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917376 GSM2917376 GEO Accession GSM2917376 SRX3545631 GSM2917377 SRP128582 SRS2819158 GSM2917377 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917377 GSM2917377 GEO Accession GSM2917377 SRX3545632 GSM2917378 SRP128582 SRS2819159 GSM2917378 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917378 GSM2917378 GEO Accession GSM2917378 SRX3545633 GSM2917379 SRP128582 SRS2819157 GSM2917379 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917379 GSM2917379 GEO Accession GSM2917379 SRX3545634 GSM2917380 SRP128582 SRS2819160 GSM2917380 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917380 GSM2917380 GEO Accession GSM2917380 SRX3545635 GSM2917381 SRP128582 SRS2819161 GSM2917381 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917381 GSM2917381 GEO Accession GSM2917381 SRX3545636 GSM2917382 SRP128582 SRS2819163 GSM2917382 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917382 GSM2917382 GEO Accession GSM2917382 SRX3545637 GSM2917383 SRP128582 SRS2819162 GSM2917383 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917383 GSM2917383 GEO Accession GSM2917383 SRX3545638 GSM2917384 SRP128582 SRS2819164 GSM2917384 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917384 GSM2917384 GEO Accession GSM2917384 SRX3545639 GSM2917385 SRP128582 SRS2819165 GSM2917385 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917385 GSM2917385 GEO Accession GSM2917385 SRX3545640 GSM2917386 SRP128582 SRS2819166 GSM2917386 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917386 GSM2917386 GEO Accession GSM2917386 SRX3545641 GSM2917387 SRP128582 SRS2819167 GSM2917387 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917387 GSM2917387 GEO Accession GSM2917387 SRX3545642 GSM2917388 SRP128582 SRS2819168 GSM2917388 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917388 GSM2917388 GEO Accession GSM2917388 SRX3545643 GSM2917389 SRP128582 SRS2819169 GSM2917389 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917389 GSM2917389 GEO Accession GSM2917389 SRX3545644 GSM2917390 SRP128582 SRS2819170 GSM2917390 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917390 GSM2917390 GEO Accession GSM2917390 SRX3545645 GSM2917391 SRP128582 SRS2819173 GSM2917391 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917391 GSM2917391 GEO Accession GSM2917391 SRX3545646 GSM2917392 SRP128582 SRS2819172 GSM2917392 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917392 GSM2917392 GEO Accession GSM2917392 SRX3545647 GSM2917393 SRP128582 SRS2819171 GSM2917393 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917393 GSM2917393 GEO Accession GSM2917393 SRX3545648 GSM2917394 SRP128582 SRS2819174 GSM2917394 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917394 GSM2917394 GEO Accession GSM2917394 SRX3545649 GSM2917395 SRP128582 SRS2819175 GSM2917395 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917395 GSM2917395 GEO Accession GSM2917395 SRX3545650 GSM2917396 SRP128582 SRS2819177 GSM2917396 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917396 GSM2917396 GEO Accession GSM2917396 SRX3545651 GSM2917397 SRP128582 SRS2819176 GSM2917397 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917397 GSM2917397 GEO Accession GSM2917397 SRX3545652 GSM2917398 SRP128582 SRS2819178 GSM2917398 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917398 GSM2917398 GEO Accession GSM2917398 SRX3545653 GSM2917399 SRP128582 SRS2819180 GSM2917399 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917399 GSM2917399 GEO Accession GSM2917399 SRX3545654 GSM2917400 SRP128582 SRS2819179 GSM2917400 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917400 GSM2917400 GEO Accession GSM2917400 SRX3545655 GSM2917401 SRP128582 SRS2819182 GSM2917401 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917401 GSM2917401 GEO Accession GSM2917401 SRX3545656 GSM2917402 SRP128582 SRS2819181 GSM2917402 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917402 GSM2917402 GEO Accession GSM2917402 SRX3545657 GSM2917403 SRP128582 SRS2819185 GSM2917403 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917403 GSM2917403 GEO Accession GSM2917403 SRX3545658 GSM2917404 SRP128582 SRS2819184 GSM2917404 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917404 GSM2917404 GEO Accession GSM2917404 SRX3545659 GSM2917405 SRP128582 SRS2819183 GSM2917405 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917405 GSM2917405 GEO Accession GSM2917405 SRX3545660 GSM2917406 SRP128582 SRS2819186 GSM2917406 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917406 GSM2917406 GEO Accession GSM2917406 SRX3545661 GSM2917407 SRP128582 SRS2819187 GSM2917407 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917407 GSM2917407 GEO Accession GSM2917407 SRX3545662 GSM2917408 SRP128582 SRS2819189 GSM2917408 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917408 GSM2917408 GEO Accession GSM2917408 SRX3545663 GSM2917409 SRP128582 SRS2819190 GSM2917409 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917409 GSM2917409 GEO Accession GSM2917409 SRX3545664 GSM2917410 SRP128582 SRS2819188 GSM2917410 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917410 GSM2917410 GEO Accession GSM2917410 SRX3545665 GSM2917411 SRP128582 SRS2819191 GSM2917411 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917411 GSM2917411 GEO Accession GSM2917411 SRX3545666 GSM2917412 SRP128582 SRS2819192 GSM2917412 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917412 GSM2917412 GEO Accession GSM2917412 SRX3545667 GSM2917413 SRP128582 SRS2819194 GSM2917413 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917413 GSM2917413 GEO Accession GSM2917413 SRX3545668 GSM2917414 SRP128582 SRS2819195 GSM2917414 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917414 GSM2917414 GEO Accession GSM2917414 SRX3545669 GSM2917415 SRP128582 SRS2819193 GSM2917415 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917415 GSM2917415 GEO Accession GSM2917415 SRX3545670 GSM2917416 SRP128582 SRS2819198 GSM2917416 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917416 GSM2917416 GEO Accession GSM2917416 SRX3545671 GSM2917417 SRP128582 SRS2819200 GSM2917417 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917417 GSM2917417 GEO Accession GSM2917417 SRX3545672 GSM2917418 SRP128582 SRS2819196 GSM2917418 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917418 GSM2917418 GEO Accession GSM2917418 SRX3545673 GSM2917419 SRP128582 SRS2819199 GSM2917419 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917419 GSM2917419 GEO Accession GSM2917419 SRX3545674 GSM2917420 SRP128582 SRS2819197 GSM2917420 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917420 GSM2917420 GEO Accession GSM2917420 SRX3545675 GSM2917421 SRP128582 SRS2819201 GSM2917421 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917421 GSM2917421 GEO Accession GSM2917421 SRX3545676 GSM2917422 SRP128582 SRS2819202 GSM2917422 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917422 GSM2917422 GEO Accession GSM2917422 SRX3545677 GSM2917423 SRP128582 SRS2819203 GSM2917423 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917423 GSM2917423 GEO Accession GSM2917423 SRX3545678 GSM2917424 SRP128582 SRS2819206 GSM2917424 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917424 GSM2917424 GEO Accession GSM2917424 SRX3545679 GSM2917425 SRP128582 SRS2819205 GSM2917425 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917425 GSM2917425 GEO Accession GSM2917425 SRX3545680 GSM2917426 SRP128582 SRS2819204 GSM2917426 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917426 GSM2917426 GEO Accession GSM2917426 SRX3545681 GSM2917427 SRP128582 SRS2819207 GSM2917427 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917427 GSM2917427 GEO Accession GSM2917427 SRX3545682 GSM2917428 SRP128582 SRS2819208 GSM2917428 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917428 GSM2917428 GEO Accession GSM2917428 SRX3545683 GSM2917429 SRP128582 SRS2819210 GSM2917429 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917429 GSM2917429 GEO Accession GSM2917429 SRX3545684 GSM2917430 SRP128582 SRS2819209 GSM2917430 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917430 GSM2917430 GEO Accession GSM2917430 SRX3545685 GSM2917431 SRP128582 SRS2819213 GSM2917431 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917431 GSM2917431 GEO Accession GSM2917431 SRX3545686 GSM2917432 SRP128582 SRS2819211 GSM2917432 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917432 GSM2917432 GEO Accession GSM2917432 SRX3545687 GSM2917433 SRP128582 SRS2819212 GSM2917433 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917433 GSM2917433 GEO Accession GSM2917433 SRX3545688 GSM2917434 SRP128582 SRS2819214 GSM2917434 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917434 GSM2917434 GEO Accession GSM2917434 SRX3545689 GSM2917435 SRP128582 SRS2819217 GSM2917435 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917435 GSM2917435 GEO Accession GSM2917435 SRX3545690 GSM2917436 SRP128582 SRS2819215 GSM2917436 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917436 GSM2917436 GEO Accession GSM2917436 SRX3545691 GSM2917437 SRP128582 SRS2819218 GSM2917437 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917437 GSM2917437 GEO Accession GSM2917437 SRX3545692 GSM2917438 SRP128582 SRS2819216 GSM2917438 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917438 GSM2917438 GEO Accession GSM2917438 SRX3545693 GSM2917439 SRP128582 SRS2819219 GSM2917439 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917439 GSM2917439 GEO Accession GSM2917439 SRX3545694 GSM2917440 SRP128582 SRS2819220 GSM2917440 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917440 GSM2917440 GEO Accession GSM2917440 SRX3545695 GSM2917441 SRP128582 SRS2819221 GSM2917441 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917441 GSM2917441 GEO Accession GSM2917441 SRX3545696 GSM2917442 SRP128582 SRS2819223 GSM2917442 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917442 GSM2917442 GEO Accession GSM2917442 SRX3545697 GSM2917443 SRP128582 SRS2819222 GSM2917443 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917443 GSM2917443 GEO Accession GSM2917443 SRX3545698 GSM2917444 SRP128582 SRS2819225 GSM2917444 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917444 GSM2917444 GEO Accession GSM2917444 SRX3545699 GSM2917445 SRP128582 SRS2819224 GSM2917445 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917445 GSM2917445 GEO Accession GSM2917445 SRX3545700 GSM2917446 SRP128582 SRS2819226 GSM2917446 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917446 GSM2917446 GEO Accession GSM2917446 SRX3545701 GSM2917447 SRP128582 SRS2819227 GSM2917447 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917447 GSM2917447 GEO Accession GSM2917447 SRX3545702 GSM2917448 SRP128582 SRS2819228 GSM2917448 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917448 GSM2917448 GEO Accession GSM2917448 SRX3545703 GSM2917449 SRP128582 SRS2819229 GSM2917449 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917449 GSM2917449 GEO Accession GSM2917449 SRX3545704 GSM2917450 SRP128582 SRS2819230 GSM2917450 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917450 GSM2917450 GEO Accession GSM2917450 SRX3545705 GSM2917451 SRP128582 SRS2819231 GSM2917451 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917451 GSM2917451 GEO Accession GSM2917451 SRX3545706 GSM2917452 SRP128582 SRS2819232 GSM2917452 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917452 GSM2917452 GEO Accession GSM2917452 SRX3545707 GSM2917453 SRP128582 SRS2819234 GSM2917453 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917453 GSM2917453 GEO Accession GSM2917453 SRX3545708 GSM2917454 SRP128582 SRS2819235 GSM2917454 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917454 GSM2917454 GEO Accession GSM2917454 SRX3545709 GSM2917455 SRP128582 SRS2819233 GSM2917455 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917455 GSM2917455 GEO Accession GSM2917455 SRX3545710 GSM2917456 SRP128582 SRS2819236 GSM2917456 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917456 GSM2917456 GEO Accession GSM2917456 SRX3545711 GSM2917457 SRP128582 SRS2819237 GSM2917457 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917457 GSM2917457 GEO Accession GSM2917457 SRX3545712 GSM2917458 SRP128582 SRS2819238 GSM2917458 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917458 GSM2917458 GEO Accession GSM2917458 SRX3545713 GSM2917459 SRP128582 SRS2819239 GSM2917459 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917459 GSM2917459 GEO Accession GSM2917459 SRX3545714 GSM2917460 SRP128582 SRS2819241 GSM2917460 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917460 GSM2917460 GEO Accession GSM2917460 SRX3545715 GSM2917461 SRP128582 SRS2819240 GSM2917461 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917461 GSM2917461 GEO Accession GSM2917461 SRX3545716 GSM2917462 SRP128582 SRS2819242 GSM2917462 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917462 GSM2917462 GEO Accession GSM2917462 SRX3545717 GSM2917463 SRP128582 SRS2819243 GSM2917463 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917463 GSM2917463 GEO Accession GSM2917463 SRX3545718 GSM2917464 SRP128582 SRS2819244 GSM2917464 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917464 GSM2917464 GEO Accession GSM2917464 SRX3545719 GSM2917465 SRP128582 SRS2819245 GSM2917465 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917465 GSM2917465 GEO Accession GSM2917465 SRX3545720 GSM2917466 SRP128582 SRS2819250 GSM2917466 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917466 GSM2917466 GEO Accession GSM2917466 SRX3545721 GSM2917467 SRP128582 SRS2819246 GSM2917467 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917467 GSM2917467 GEO Accession GSM2917467 SRX3545722 GSM2917468 SRP128582 SRS2819247 GSM2917468 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917468 GSM2917468 GEO Accession GSM2917468 SRX3545723 GSM2917469 SRP128582 SRS2819248 GSM2917469 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917469 GSM2917469 GEO Accession GSM2917469 SRX3545724 GSM2917470 SRP128582 SRS2819249 GSM2917470 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917470 GSM2917470 GEO Accession GSM2917470 SRX3545725 GSM2917471 SRP128582 SRS2819251 GSM2917471 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917471 GSM2917471 GEO Accession GSM2917471 SRX3545726 GSM2917472 SRP128582 SRS2819252 GSM2917472 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917472 GSM2917472 GEO Accession GSM2917472 SRX3545727 GSM2917473 SRP128582 SRS2819253 GSM2917473 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917473 GSM2917473 GEO Accession GSM2917473 SRX3545728 GSM2917474 SRP128582 SRS2819254 GSM2917474 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917474 GSM2917474 GEO Accession GSM2917474 SRX3545729 GSM2917475 SRP128582 SRS2819255 GSM2917475 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917475 GSM2917475 GEO Accession GSM2917475 SRX3545730 GSM2917476 SRP128582 SRS2819256 GSM2917476 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917476 GSM2917476 GEO Accession GSM2917476 SRX3545731 GSM2917477 SRP128582 SRS2819259 GSM2917477 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917477 GSM2917477 GEO Accession GSM2917477 SRX3545732 GSM2917478 SRP128582 SRS2819258 GSM2917478 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917478 GSM2917478 GEO Accession GSM2917478 SRX3545733 GSM2917479 SRP128582 SRS2819257 GSM2917479 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917479 GSM2917479 GEO Accession GSM2917479 SRX3545734 GSM2917480 SRP128582 SRS2819260 GSM2917480 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917480 GSM2917480 GEO Accession GSM2917480 SRX3545735 GSM2917481 SRP128582 SRS2819261 GSM2917481 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917481 GSM2917481 GEO Accession GSM2917481 SRX3545736 GSM2917482 SRP128582 SRS2819262 GSM2917482 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917482 GSM2917482 GEO Accession GSM2917482 SRX3545737 GSM2917483 SRP128582 SRS2819263 GSM2917483 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917483 GSM2917483 GEO Accession GSM2917483 SRX3545738 GSM2917484 SRP128582 SRS2819264 GSM2917484 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917484 GSM2917484 GEO Accession GSM2917484 SRX3545739 GSM2917485 SRP128582 SRS2819265 GSM2917485 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917485 GSM2917485 GEO Accession GSM2917485 SRX3545740 GSM2917487 SRP128582 SRS2819266 GSM2917487 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917487 GSM2917487 GEO Accession GSM2917487 SRX3545741 GSM2917490 SRP128582 SRS2819267 GSM2917490 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917490 GSM2917490 GEO Accession GSM2917490 SRX3545742 GSM2917494 SRP128582 SRS2819268 GSM2917494 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917494 GSM2917494 GEO Accession GSM2917494 SRX3545743 GSM2917497 SRP128582 SRS2819270 GSM2917497 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917497 GSM2917497 GEO Accession GSM2917497 SRX3545744 GSM2917500 SRP128582 SRS2819269 GSM2917500 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917500 GSM2917500 GEO Accession GSM2917500 SRX3545745 GSM2917503 SRP128582 SRS2819271 GSM2917503 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917503 GSM2917503 GEO Accession GSM2917503 SRX3545746 GSM2917506 SRP128582 SRS2819272 GSM2917506 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917506 GSM2917506 GEO Accession GSM2917506 SRX3545747 GSM2917509 SRP128582 SRS2819273 GSM2917509 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917509 GSM2917509 GEO Accession GSM2917509 SRX3545748 GSM2917511 SRP128582 SRS2819274 GSM2917511 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917511 GSM2917511 GEO Accession GSM2917511 SRX3545749 GSM2917513 SRP128582 SRS2819275 GSM2917513 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917513 GSM2917513 GEO Accession GSM2917513 SRX3545750 GSM2917514 SRP128582 SRS2819276 GSM2917514 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917514 GSM2917514 GEO Accession GSM2917514 SRX3545751 GSM2917515 SRP128582 SRS2819277 GSM2917515 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917515 GSM2917515 GEO Accession GSM2917515 SRX3545752 GSM2917516 SRP128582 SRS2819278 GSM2917516 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917516 GSM2917516 GEO Accession GSM2917516 SRX3545753 GSM2917517 SRP128582 SRS2819279 GSM2917517 RNA-Seq TRANSCRIPTOMIC cDNA Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified. Illumina HiSeq 2500 gds 302917517 GSM2917517 GEO Accession GSM2917517