 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 06, 2018 |
| Title |
Ad-Id4GFP-Seq1_C81 |
| Sample type |
SRA |
| |
|
| Source name |
Adult testis
|
| Organism |
Mus musculus |
| Characteristics |
egfp epifluorescence quartile: Q2
cell type: Spermatogonium
|
| Treatment protocol |
Animal were untreated.
|
| Growth protocol |
Normal mouse husbandry with ad libitum food and water.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single-cell suspensions of seminiferous tubules were generated from adult mouse testes using a two-step enzymatic digestion approach. Briefly, testicular parenchyma from 2 or more adult mice were digested with 1mg/ml Collagenase Type IV (Worthington Biochemicals) for 2-3 minutes at 37oC, washed with Hank’s Buffered Salt Solution (HBSS) to remove interstitial cells, digested with 0.25% trypsin/EDTA containing 1.4mg/ml DNase I (Sigma) for 7-9 minutes at 37oC, and quenched with 10% FBS. Testis cell suspensions were used for FACS to enrich spermatogonia essentially as described (Hermann et al., 2015). Briefly, cells were suspended (5-20 x 10^6 cells/ml) in ice-cold Dulbecco’s phosphate-buffered saline (DPBS) containing 10% FBS (DPBS+S). Cells were pre-enriched for spermatogonia by density centrifugation in DPBS+S over a 30% Percoll cushion (Sigma) for 8 minutes at 600xg without braking. Pelleted cells were subjected to FACS and spermatogonia isolated based on gating CD9-bright and ID4-EGFP double-positive cells using a FACS Aria (BD). CD9bright/ID4-EGFP+ spermatogonia adult mice were used for single-cell RNA-seq facilitated by the Fluidigm C1 instrument essentially as described (Hermann et al., 2015; Wu et al., 2014). Briefly, single cells were captured on 10-17µm integrated fluidic circuit chips using the C1 Single-Cell Autoprep System (Fluidigm), stained with ethidium homodimer, and imaged on an AxioImager M1 (Zeiss), and used to prepare cDNA with SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (v2 chemistry; Takara). Dead cells (ethidium+), multiplets and cells contaminated with debris were excluded from further analysis. Mouse ID4-EGFP+ spermatogonia were stratified based on the EGFP epifluorescence intensity using the interactive measurement module of AxioVision 4.8.2 (Zeiss) and images taken of each cell. The densitometric mean value of the EGFP channel for each cell was normalized on a scale from 0 to 1 (1 representing the brightest EGFP+ cell) and cells were ranked. ID4-EGFPbright and ID4-EGFPdim spermatogonia were defined as the upper (Q1) or lower (Q2) quartiles (25%) of ranked EGFP+ cells, respectively.
Amplified cDNA was quantified with PicoGreen fluorometry (ThermoFisher Scientific) on a Synergy II (Biotek) based on manufacturer recommendations and normalized cDNA mass from each cell was used for Nextera XT dual-index library preparation (Illumina) with modifications from manufacturer recommendations essentially as described (Mutoji et al., 2016). Single-cell libraries were pooled, qualified for fragment size and distribution on a 2100 Bioanalyzer (Agilent) and quantified.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Ad-Id4GFP-C1_GEmatrix
|
| Data processing |
Illumina base-calling (PE100) and demultiplexing with BCL-2-Fastq
Paired FASTQ files from each sample trimmed and the quality confirmed using FASTQC
Aligned to the mouse genome (mm10) with TopHat (Galaxy v0.9)
Transcript abundance (FPKM) determined with Cufflinks (Galaxy v.2.2.1.0)
Quality control with the Picard tool Collectrnaseqmetrics was used to eliminate poorly performing cell samples
Genome_build: Mouse reference genome NCBI build 38, GRCm38 (mm10)
Supplementary_files_format_and_content: A single matrix file CSV containing columns of FPKM values for each cell. Rows are FPKM values for each gene in the reference annotation. First column is gene ID, first row is sample name.
|
| |
|
| Submission date |
Jan 09, 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 |
GPL17021 |
| Series (1) |
| GSE108974 |
C1 single-cell RNA-seq of Adult ID4-EGFP mouse spermatoognia |
|
| Relations |
| BioSample |
SAMN08333602 |
| SRA |
SRX3545510 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
