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Status |
Public on Nov 06, 2018 |
Title |
AdultHuman-Spermatids_17-6 |
Sample type |
SRA |
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Source name |
Adult testis
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Organism |
Homo sapiens |
Characteristics |
number of cells: 3086 cell type: Sta-Put enriched Spermatids
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Treatment protocol |
De-identified, surgical excess normal adult human testicular tissue was obtained through the Infertility Center of St. Louis from patients undergoing a diagnostic testicular biopsy (n=28) in preparation for microscopic vasectomy reversal (MVR) or testicular sperm extraction (TESE) for obstructive azoospermia with the informed consent of patients (see Supplemental Table 2). Testicular tissue was also recovered from male organ donors (n=2) through the Texas Organ Sharing Alliance and the University of Texas Transplant Center with informed consent of the next of kin. The University of Texas at San Antonio IRB determined that research on these tissues did not constitute human subjects research (IRB #12-098N and #17-074N). The age of the individuals was recorded (median = 42yr, mean = 41.6 ± 1.3yr), and while organ donors tended to be younger (34.3 ± 7.2yr) than biopsy patients (42.4 ± 1.2yr), the difference was not significant (p=0.32). Procured tissue was transported to the laboratory on ice in either Lactated Ringer’s solution or minimal essential medium alpha (MEMα) containing 10% fetal bovine serum. The amount of time tissues were exposed to cold ischemia prior to processing was recorded (median = 18.7hr; mean = 24.7 ± 3.8 hr).
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Growth protocol |
N/A
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Extracted molecule |
polyA RNA |
Extraction protocol |
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). Adult Human testis cells were also enriched for spermatocytes and spermatids based on sedimentation velocity at unit gravity (Romrell et al., 1976; Bellve et al., 1977). Briefly, testis cells (10^6-10^7) suspended in 2 ml of buffer plus 0.5% BSA were loaded onto a 50 ml gradient of 2-4% BSA (McCarrey et al., 1992) and allowed to sediment for 2.5 hr at 4°C. Approximately one hundred 0.5 ml fractions were then collected in microcentrifuge tubes and analyzed for content of spermatocytes or spermatids on the basis of morphology under phase contrast optics (which typically yields ≥85% purity). Fractions containing spermatocytes or spermatids were pooled separately, concentrated (to ~2 x106 cells/ml) and stored in buffer containing FBS on ice until use. Ultimately, sorted spermatoginia,Sta-Put-enriched spermatocytes and spermatids, or unselected spermatogenic cells from adult human testicular tissue were used for single-cell RNA-seq facilitated by the 10X Genomics Chromium Drop-Seq approach. Briefly, Cell suspensions were loaded into Chromium microfluidic chips with 3’ v2 chemistry and used to generate single-cell gelbead emulsions (GEMs) with using the Chromium controller (10X Genomics) per manufacturer recommendations (Zheng et al., 2017). In all cases, suspensions containing ~8700 cells were loaded on the instrument with the expectation of collecting up to 5,000 GEMs containing single cells. For the multiplet test (Fig. S1), equal numbers of mouse and human cells were loaded. GEM-RT was performed in a T100 Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to manufacturer recommendations. Single-cell 3' libraries were prepared according to manufacturer recommendations (v2 chemistry).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
single-cell
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Data processing |
Illumina base-calling and demultiplexing with BCL-2-Fastq. Trimmed FASTQ files (26bp Cell barcode and UMI Read1, 8bp i7 index, and 100bp Read2), were generated using the CellRanger mkfastq command (a 10X Genomics wrapper around BCL2Fastq). Primary data analysis (alignment, filtering, and UMI counting) to determine gene transcript counts per cell (producing a gene-barcode matrix), quality control, clustering and statistical analysis were performed using CellRanger count (10X Genomics) and the hg19 (human), mm10 (mouse) or hg19+mm10 (human and mouse, for multiplet tests) genome assembly/annotation references Outputs from multiple independent samples of single-cells were combined using CellRanger aggr (10X Genomics) based on mapped read counts to normalize sequencing depth and produce aggregated gene-barcode matrices and clustering models. Genome_build: Human reference genome NCBI build 38 (GRCh38) or a combined hg19 (hGRC37) + Mouse reference genome NCBI build 38, GRCm38 (mm10) for multiplet tests Supplementary_files_format_and_content: A single gene x. cell-barcode matrix file (MTX). Each matrix is stored in Market Exchange Format (MEX). It also contains TSV files with genes and barcode sequences corresponding to row and column indices, respectively.
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Submission date |
Jan 10, 2018 |
Last update date |
Nov 06, 2018 |
Contact name |
Brian P. Hermann |
E-mail(s) |
Brian.Hermann@utsa.edu
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Phone |
210-458-8047
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Organization name |
University of Texas at San Antonio
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Department |
Department of Biology
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Street address |
1 UTSA Circle
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City |
San Antonio |
State/province |
TX |
ZIP/Postal code |
78249 |
Country |
USA |
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Platform ID |
GPL18573 |
Series (1) |
GSE109037 |
10x Genomics Drop-seq single-cell RNA-seq of isolated Adult human spermatogonia, spermatocytes, spermatids & steady-state spermatogenic cells |
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Relations |
BioSample |
SAMN08347950 |
SRA |
SRX3549441 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2928384_AdultHuman-Spermatids_17-6_barcodes.tsv.gz |
13.2 Kb |
(ftp)(http) |
TSV |
GSM2928384_AdultHuman-Spermatids_17-6_genes.tsv.gz |
258.6 Kb |
(ftp)(http) |
TSV |
GSM2928384_AdultHuman-Spermatids_17-6_matrix.mtx.gz |
41.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
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