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Sample GSM4253857

Query DataSets for GSM4253857

Status

Public on Sep 18, 2020

Title

065_vivo-comp-GV-2_rep3

Sample type

SRA

Source name

vivo-comp-GV-2_rep3

Organism

Mus musculus

Characteristics

strain: C57BL/6 X 129
genotype: Blimp1-Venus and Stella-CFP
cell type: in vivo oocytes

Growth protocol

ESC lines were cultured in 2i plus LIF without feeders (Ying et al., 2008). PGCLC induction was performed as described (Hayashi and Saitou, 2013). PGCLCs were purified by FACSAria II or Fusion (BD Bioscience) and aggregated with E12.5 female gonadal somatic cells in a low-binding U-bottom 96-well plate (NUNC) for 2 days of culture in GK15 supplemented with 1 μM retinoic acid. To strictly remove residual PGCs from dissociated gonadal cells, both SSEA1 and CD31 antibodies (Miltenyi Biotech) were used according to the manufacturer’s instructions. 5,000 PGCLCs were cultured with 75,000 gonadal somatic cells to produce one reconstituted ovary. IVD culture was performed as described (Hikabe et al., 2016). rOvaries (5,000 PGCLCs and 75,000 gonadal somatic cells) were placed on Transwell-COL membranes soaked in αMEM-based IVDi medium: αMEM supplemented with 2% FCS, 150 μM ascorbic acid (Sigma), 1× Glutamax, 1× penicillin/streptomycin and 55 μM 2-mercaptoethanol (Life Technologies). At 4 days of culture, the culture medium was changed to StemPro-34-based IVDi medium: StemPro-34 SFM (Life Technologies) supplemented with 10% FCS, 150 μM ascorbic acid, 1× Glutamax, 1× penicillin/streptomycin, and 55 μM 2-mercaptoethanol. From 7 days to 10 days of culture, 500 nM ICI182780 was added to the StemPro-34-based IVDi medium. IVG culture was performed as described (Hikabe et al., 2016). Briefly, the isolated secondary follicles on the Transwell-COL membranes were soaked in IVG medium: αMEM supplemented with 5% FCS, 2% polyvinylpyrrolidone (Sigma), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin/streptomycin, 100 μM 2-mercaptoethanol, 55 μg ml−1 sodium pyruvate (Nacalai Tesque), 0.1 IU ml−1 follicle-stimulating hormone (Follistim; MSD), 15 ng ml−1 BMP15 and 15 ng ml−1 GDF9 (R&D Systems). At 2 days of culture, BMP15 and GDF9 were withdrawn from the medium and then follicles were incubated in 0.1% TypeIV Collagenase (MP Biomedicals). After washing with αMEM supplemented with 5% FCS several times, the follicles were cultured in IVG-αMEM without BMP15 and GDF9. At 11 days of culture, cumulus-oocyte complexes grown on the membrane were picked up by a fine glass capillary. Cumulus–oocyte complexes were transferred to IVM medium: αMEM supplemented with 5% FCS, 25 μg ml−1 sodium pyruvate, 1× penicillin/streptomycin, 0.1 IU ml−1 follicular-stimulating hormone, 4 ng ml−1 EGF, and 1.2 IU ml−1 hCG (gonadotropin; ASKA). At 16 h of culture, swollen cumulus cells were stripped from the oocytes by treating with hyaluronidase (Sigma), and then MII oocytes were determined by 1st polar body extrusion. For direct induction of ngDIOs, 5x10^4 ESCs were transferred into a low-cell-binding U-bottom 96-well plate (NUNC) in S10 medium (StemPro-34 SFM (Life Technologies) supplemented with 10% FCS, 150 μM ascorbic acid, 1× Glutamax, 1× penicillin/streptomycin and 55 μM 2-mercaptoethanol) with 150 ng/ml SCF, 0.5 µM of Shield1 (Clontech), and 10 nM Y-27632 (Wako). For the extended culture, we transferred the aggregates onto Transwell-COL membranes using mouth pipet on day 2, and cultured under normoxic conditions (20% O2 and 5% CO2 at 37 °C) for 2-4 weeks. For induction of GV and MII DIO, 5x10^4 ESCs were aggregated with 30,000-75,000 E12.5 female gonadal somatic cells in S10 medium with 0.5 µM of Shield1 and cultured for 2 days and transferred onto Transwell-COL membranes (Corning) soaked in S10 medium with 0.5 µM of Shield1 and cultured for 28 days. At 21-28 days of culture, individual follicles were manually isolated using sharpened tungsten needles. The Isolated follicles were further differentiated by IVG for 7-11 days and IVM culture and fertilized by IVF as previously described (Hayashi et al., 2017; Hikabe et al., 2016).

Extracted molecule

polyA RNA

Extraction protocol

IVD culture was performed as described previously (Hikabe et al., 2016, Nature). rOvaries (5,000 PGCLCs and 75,000 gonadal somatic cells) were placed on Transwell-COL membranes soaked in αMEM-based IVDi medium: αMEM supplemented with 2% FCS, 150 μM ascorbic acid (Sigma), 1× Glutamax, 1× penicillin/streptomycin and 55 μM 2-mercaptoethanol (Life Technologies). At 4 days of culture, the culture medium was changed to StemPro-34-based IVDi medium: StemPro-34 SFM (Life Technologies) supplemented with 10% FCS, 150 μM ascorbic acid, 1× Glutamax, 1× penicillin/streptomycin, and 55 μM 2-mercaptoethanol. From 7 days to 10 days of culture, 500 nM ICI182780 was added to the StemPro-34-based IVDi medium.Ovaries (in vivo) and rOvaries (in vitro) at each developmental stage were treated with CTK solution (0.1 mg/ml collagenase IV, 0.25% trypsin, 20% KSR and 1 mM CaCl2 in PBS) for 30 minutes at 37°C, then transferred into Accutase (Nacalai Tesque) and incubated at 37°C for 15 minutes. The IVD day11 or day13 SC+oocytes were isolated by FACS-sorting. For FACS preparation, the cells were dissociated by gentle pipetting and filtered by using a 70 μm-pored nylon mesh to remove cell clumps.
Ovaries (in vivo) and rOvaries (in vitro) at each developmental stage were treated with CTK solution (0.1 mg/ml collagenase IV, 0.25% trypsin, 20% KSR and 1 mM CaCl2 in PBS) for 30 minutes at 37°C, then transferred into Accutase (Nacalai) and incubated at 37°C for 15 minutes. The IVD day11 or day13 SC+oocytes were isolated by FACS-sorting. For FACS preparation, the cells were dissociated by gentle pipetting and filtered by using a 70 μm-pored nylon mesh to remove cell clumps. Collected samples were transferred into a 1.5 ml DNA LoBind tube (Eppendorf). Directional RNA-seq libraries were prepared as described previously (Hamazaki et al., 2015). Briefly, polyA-tailed RNA were extracted by Dynabeads mRNA Purification Kit.
Extracted polyA+RNA were converted to cDNA libraries by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Fastq files from sequencing experiments were mapped to the mouse mm10 genome using default parameters for STAR (v2.6.0, Dobin et al., 2013)
Mapped reads were counted by featureCount (v1.6.0, Liao et al., 2014, -O -s 2).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files including TPM values for each Sample.

Submission date

Jan 07, 2020

Last update date

Sep 18, 2020

Contact name

Nobuhiko Hamazaki

E-mail(s)

hamazaki@hgs.med.kyushu-u.ac.jp

Organization name

Kyushu University

Department

Department of Developmental Stem Cell Biology