Illumina HiSeq 2500 paired end sequencing; GSM2917200: Ad-Id4GFP-Seq1_C01; Mus musculus; RNA-Seq

Ad-Id4GFP-Seq1_C01

To reveal distinct transcriptomes associated with spermatogonial stem cell renewal vs. initiation of differentiation, single-cell transcriptomes from Adult ID4-EGFP+ spermatogonia were subdivided into subpopulations that displayed distinct fates 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: Four replicate preparations of Adult mouse ID4-EGFP+ spermatogonia were used for this study. Data are from a total of 300 cells. Cells were binned into quartiles according to EGFP epifluorescence intensity (Q1 = EGFP-bright, Q4=EGFP-dim, Q2 and Q3 have intermediate EGFP fluorescence) to facilitate comparisons.

C1 single-cell RNA-seq of Adult ID4-EGFP mouse spermatoognia

Submitted by Gene Expression Omnibus on 07-NOV-2018

Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank's Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco's phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using  a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17Âµm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively. Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially 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 ID4-EGFP mouse spermatoognia

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