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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 30, 2021 |
| Title |
E18.5_2 |
| Sample type |
SRA |
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| Source name |
Prospermatogonia
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| Organism |
Mus musculus |
| Characteristics |
genotype: Ddx4-RFPTg/0
cell type: Germ cells
strtain: ICR x B6C3(Cg)B6F1(5N)
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| Treatment protocol |
Isolation of Germ Cells To isolate germ cells for the RNA-seq analysis, ICR females were crossed with C57BL/6N; mvh-RFPTg/Tg males. The embryonic gonads/postnatal testis fragments at E12.5–P7.5 were dissociated into single cells by incubating with 0.05% Trypsin-EDTA (GIBCO, #15400-054) for 10 min at 37℃, and were quenched with an equal volume of DMEM medium (GIBCO, #10313-021) containing 10% FCS (Hyclone), 2 mM GlutaMAX (GIBCO, #35050-061), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO, #15070). Large clumps of cells were removed using a nylon cell strainer (FALCON, #352350), and cells were centrifuged at 1,200 rpm for 5 min. The cell pellet was re-suspended with DMEM/F12 medium (GIBCO, 1130-082) containing 0.1% BSA fraction V (Life Technologies, # 15260037), and sorted with a flow cytometer (BD Biosciences, FACS Aria Ⅲ) Fluorescence-activated Cell Sorting (FACS) The d4 floating aggregates of EpiLCs and d4c5 mPGCLCs were incubated in TrypLE express for 10 min at 37℃, with periodical pipetting, and the dissociates were quenched with a 5 × volume of DMEM/F12 medium (GIBCO, #1130-082) containing 0.1% BSA fraction V (Life Technologies, # 15260037) and DNase I (Sigma, DN25), followed by pipetting to generate a single cell suspension. After centrifugation at 1,200 rpm for 5 min, the supernatant was discarded. The cell pellet was re-suspended with DMEM/F12 medium (GIBCO, #1130-082) containing 0.1% BSA fraction V (Life Technologies, #15260037), the cell suspension was filtered through a nylon cell strainer (FALCON, #352350), and BV+ d4 mPGCLCs or BV+; SC+ d4c5 mPGCLCs were sorted by a flow cytometer (BD Biosciences, FACSAria III) and collected in the GK10+FR10 medium. The rTestes at various culture periods were peeled off from the cell insert membrane using a P1000 pipette tip and incubated in 0.25% Trypsin-EDTA in PBS (GIBCO, #15400-054) for 5~15 min, with periodical intermittent pipetting, and the dissociates were quenched by an equal volume of DMEM medium (GIBCO, #10313-021) containing 10% FCS (Hyclone), 2 mM GlutaMAX (GIBCO, 35050-061), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO, 15070). The resultant cell pellet was resuspended with PBS containing 0.1% BSA fraction V (Life Technologies, # 15260037), and large clumps of cells were removed using a nylon cell strainer (FALCON, #352350). The BV+; SC+ and Ddx4/mvh-RFP-positive (BV+; SC+; VR+) cells (mPGCLC-derived cells in rTestes: m0, m3, m5, m7, m10), VR+ and CD9-APC-positive (VR+; CD9-APC+) cells (mPGCLC-derived cells in rTestes: m12, m14), and VR+ cells in embryonic gonads/postnatal testes were sorted by the FACSAria III (BD Biosciences) cell sorter, and were collected in CELLOTION (Nippon Zenyaku Kogyo, CB051). For RNA/DNA extraction, the sorted cells were centrifuged at 500G for 10 min, and the cell pellets were frozen down by liquid N2, and preserved at −80℃.
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| Growth protocol |
Generation and Culture of Reconstituted Testes (rTestis) To isolate E12.5 mouse male gonads (fetal testes), pregnant ICR females were sacrificed by cervical dislocation, and the embryos were isolated in chilled Dulbecco's Modified Eagle Medium (DMEM) (GIBCO, #10313-021) containing 10% FCS (Hyclone), 2 mM GlutaMAX (GIBCO, #35050-061), 10 mM HEPES (GIBCO, #15630-106), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO, 15070). Fetal testes were identified by their appearance, and the mesonephroi were dissected out with tungsten needles. The isolated fetal testes were dissociated into single cells, and endogenous mouse germ cells were selected out by magnetic-activated cell sorting (MACS). Compared to FACS, MACS requires much less time and less physical stress to collect the cells for the rTestis formation. The mPGCLCs (10,000 cells/well) and the fetal testes somatic cells (when using frozen stocks, 20,000 cells/well) (total 30,000 cells/well) were mixed and plated on the wells of a Nunclon Sphera 96-well Microplate (#174925; Thermo Fisher Scientific) and cultured under a floating condition in GK10+FR10 medium (GK10, 10 μM forskolin and 10 μM rolipram). After a period of 2 days for the floating culture, using a glass capillary, the rTestes were transferred onto ThinCert™Cell Culture Inserts for 24-Well Plates (Greiner #662610) soaked in alpha-Minimum Essential Medium (GIBCO, #32561-037) containing 10% FCS (Hyclone), 55 μM 2-mercaptoethanol (GIBCO, 21985-023), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO, 15070). Half the medium was changed every two days. All rTestes were cultured for a gas-liquid interphase culture at 37℃ under an atmosphere of 5% CO2 in air. Derivation of GSCLCs from rTestis For the derivation of GSCLCs, VR+; CD9-APC+ cells in rTestes (m14) were sorted with a flow cytometer (BD Biosciences, FACS Aria Ⅲ). For deriving GSCLCs from all VR+; CD9-APC+ cells/rTestis, 24-well culture dishes (Falcon, #353047) were coated with PBS supplemented with 0.1% gelatin for at least 30 min, seeded with mouse embryonic feeders (MEF) for at least 6 h prior to the plating of mPGCLC-derived cells, and then VR+; CD9-APC+ cells (derived from 1 rTestis)/well were seeded and cultured with GSCs/GSCLCs medium. For deriving GSCLCs from single VR+; CD9-APC+ cells, 96-well culture dishes (Falcon, #353072) were coated with PBS supplemented with 0.1% gelatin for at least 30 min, then seeded with MMC-treated MEF for at least 6 h prior to the plating of an mPGCLC-derived cell. A single VR+; CD9-APC+ cell was then seeded using a FACS Automated Cell Deposition Unit (ACDU) system and cultured with GSCs/GSCLCs medium.The GSC/GSCLC medium was as described previously (Kanatsu-Shinohara et al., 2003), with some modifications (Ishikura et al., 2016). StemPro-34 SFM (GIBCO, #10639011) was supplemented with StemPro supplement (GIBCO, #10639011), 0.1 mM 2-mercaptoethanol (GIBCO, 21985-023), 1% FCS (Hyclone), 1 × minimal essential medium (MEM) Vitamin Solution (GIBCO, # 11120-052), 5.0 mg/mL AlbuMAXⅠ (GIBCO, #11020-021), 0.1 mM NEAA (GIBCO, #11140-050), 1 mM sodium pyruvate (GIBCO, 11360-070), 100 U/mL penicillin, 0.1 mg/mL streptomycin (GIBCO, #15070), 2 mM GlutaMAX (GIBCO, #35050-061), 1 × Insulin-Transferrin-Selenium (ITS-G) (GIBCO, #41400045), 10 ng/mL bFGF Recombinant Human Protein (Life Technologies, #13256029), 20 ng/mL GDNF Rat Recombinant (RSD, #512GF050), 20 ng/mL EGF Mouse Recombinant Carrier-free (RSD, #2028EG200), and 1000 U/mL LIF (Merk, ESG1107). Half the medium was changed every two or three days. All GSCs/GSCLCs were cultured at 37℃ under an atmosphere of 5% CO2 in air.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were lysed and the total RNAs were purified using a NucleoSpin RNA XS (Takara Bio, #U0902A) according to the manufacturer’s instructions. 1 ng of total RNAs from each sample was used for the synthesis and amplification of cDNAs.
The cDNA synthesis was performed according to the single-cell 3 prime RNA-seq (SC3-seq) method described previously (Nakamura et al., 2015). The cDNA libraries for Nextseq500 (Illumina) were constructed as described previously (Ishikura et al., 2016).The sequencing was performed on an Illumina Nextseq500/550 platform with a Nextseq500/550 Hiqh output kit (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
3'-RNA-seq amplified from 1ng RNA
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| Data processing |
The processing of the 3-prime-sequencing data for calculating the gene-expression levels was performed as described previously (Nakamura et al., 2015). In brief, all reads were processed with cutadapt v1.9.1 to remove adaptors and poly-A sequences.
The reads of 30-bp or longer were mapped onto a mouse transcript reference, ref_GRCm38.p6, using Tophat v2.0.11/Bowtie2 v2.2.7. Read counts per gene were calculated using HTSeq v0.9.1 with the default settings. Read counts were converted to read counts per million mapped reads (RPM) and used for the analysis.
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: Tab-delimited text files include RPM values for each sample.
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| Submission date |
Mar 04, 2021 |
| Last update date |
Sep 30, 2021 |
| Contact name |
Yukihiro Yabuta |
| E-mail(s) |
yabyab@anat2.med.kyoto-u.ac.jp
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| Organization name |
Kyoto University, Graduate school of medicine
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| Department |
Anatomy and Cell Biology
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| Street address |
Yoshida-Konoe-cho, Sakyo-ku
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| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
606-8501 |
| Country |
Japan |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE168220 |
In Vitro Reconstitution of the Whole Male Germ-Cell Development from Mouse Pluripotent Stem Cells [RNA-Seq] |
| GSE168222 |
In Vitro Reconstitution of the Whole Male Germ-Cell Development from Mouse Pluripotent Stem Cells |
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| Relations |
| BioSample |
SAMN18139173 |
| SRA |
SRX10237424 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5134162_rnaseq_E18_5_2_RPM.txt.gz |
334.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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