Illumina HiSeq 2500 paired end sequencing; GSM2917543: HumanSpg-Seq1_C01; Homo sapiens; RNA-Seq

HumanSpg-Seq1_C01

To reveal distinct transcriptomes associated with spermatogonial stem cell renewal vs. initiation of differentiation, single-cell transcriptomes from Adult Human spermatogonia were subdivided into subpopulations based on the levels of ID4 mRNA (determined in this experiment). This correlates with distinct fates of corresponding mouse spermatogonia when assayed by transplantation, with ID4-EGFPbright cells highly enriched for SSCs, and ID4-EGFPdim cells enriched for progenitors. We used the Fluidigm C1 instrument to capture individual spermatogonia for SMART-Seq2 single-cell RNA-seq. Overall design: Nine replicate preparations of Adult Human spermatogonia were used for this study. Data are from a total of 635 cells. Cells were binned into quartiles according to ID4 mRNA levels (Q1 = ID4-high, Q4=ID4-low, Q2 and Q3 have intermediate ID4 mRNA levels) to facilitate comparisons.

C1 single-cell RNA-seq of Adult Human spermatoognia

Submitted by Gene Expression Omnibus on 07-NOV-2018

Suspensions of human seminiferous tubule cells were prepared from adult testicular tissue parenchyma by a two-step enzymatic digestion was as described previously (Hermann et al., 2007b; Hermann et al., 2009; Dovey et al., 2013). Briefly, testis tissue was digested with 2mg/ml collagenase type IV at 37oC with vigorous agitation, washed with HBSS to remove interstitial cells, followed by digestion with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I for 15 minutes at 37oC with trituration every 5 minutes, and quenched with 10% FBS. In all cases, cell suspensions were strained (40um pore size for mouse, 70um pore size for humans) and suspended in MEMα containing 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 106 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S), labeled with antibodies (see Key Resources Table), and subjected to FACS using  a FACS Aria (BD). Positive antibody labeling was determined by comparison to staining with isotype control antibodies. Human spermatogonia were isolated by sorting cells with the phenotype HLA-ABCnegative / CD49enegative / THY1dim / ITGA6+ / EpCAMdim dim (see Fig. 2C). These markers were chosen because previous results indicate they specifically label undifferentiated spermatogonia, including some with colonization potential based on xenotransplantation (Dovey et al., 2013; Altman et al., 2014; Smith et al., 2014; Valli et al., 2014). Human spermatogonia were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Human spermatogonia were stratified post-hoc based on the levels of D4 mRNA to match EGFP epifluorescence intensity stratification used for cognate moues cells. ID4 mRNA levels were normalized on a scale from 0 to 1 (1 representing the greatest amount of ID4mRNA) and cells were ranked. ID4-high and ID4-low spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified.

C1 single-cell RNA-seq of Adult Human spermatoognia

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