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Sample GSM3302524 Query DataSets for GSM3302524
Status Public on Nov 06, 2018
Title AdultHuman-Spermatocytes_17-11
Sample type SRA
 
Source name Adult testis
Organism Homo sapiens
Characteristics number of cells: 2127
cell type: Sta-Put enriched Spermatocytes
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. 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).
Growth protocol N/A
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. 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, 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).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description single-cell
AdultHuman-Spermatocytes_17-11_barcodes.tsv
AdultHuman-Spermatocytes_17-11_genes.tsv
AdultHuman-Spermatocytes_17-11_matrix.mtx
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.
 
Submission date Jul 23, 2018
Last update date Nov 06, 2018
Contact name Brian P. Hermann
E-mail(s) Brian.Hermann@utsa.edu
Phone 210-458-8047
Organization name University of Texas at San Antonio
Department Department of Biology
Street address 1 UTSA Circle
City San Antonio
State/province TX
ZIP/Postal code 78249
Country USA
 
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
Relations
BioSample SAMN09704209
SRA SRX4419805

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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