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| Status |
Public on Aug 16, 2024 |
| Title |
C25 |
| Sample type |
SRA |
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| Source name |
ovarian follicle
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| Organism |
Homo sapiens |
| Characteristics |
tissue: ovarian follicle
cell line: iPSC-derived MCP
disease: Control
treatment: No
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| Treatment protocol |
The STEMdiff mesenchymal progenitor kit (STEMCELL) was utilized to differentiate MPCs. 2 days before induction, iPSCs were cultured without feeder cells and exposed to 10 μM Y-27632 for 2 h. After 2 h, the iPSCs dissociated into single cells using TypLE, then replating at a concentration of 5×104 cells/cm2 in Essential 8 medium with 10 μM Y-27632. The fresh medium was replaced the following day. From the start of day 0 until day 3, the medium was switched to the mesenchymal induction medium. The medium was changed to MesenClut-ACF medium on day 4. On day 6, these cells were exposed to 10 μM Y-27632 for 2 h and dissociated using TypLE, followed by plating at a concentration of 3-10x103 cells/cm2 in MesenClut-ACF medium MesenClut-ACF attachment substrate-coated plates. Once the cells reached 80% confluency, 10-7 M testosterone was added for three weeks, with the medium being changed weekly.
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| Growth protocol |
Follicular aspirates were collected during oocyte retrieval from both healthy control and PCOS patients undergoing in vitro fertilization for infertility treatment. Granulosa cells in follicular aspirates were isolated by using Ficoll density gradient centrifugation. The cells were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum, 1% MEM non-essential amino acids (NEAA), 1% GlutaMAX, and 1% antibiotic-antimycotic. Infection was preceded by 2 days of seeding 2-5×105 granulosa cells in a 35 mm dish. The virus infection procedures followed the manufacturer's instructions (Invitrogen) and the medium was replaced with fresh medium daily. One week after infection, the cells were collected using Accumax (eBioscience) and replated on mitomycin C-inactivated mouse embryo fibroblasts as feeder cells. On the next day, the medium was changed to DMEM/F12 medium supplemented with 20% knockout serum replacement (KOSR), 1% NEAA, 0.5% GlutaMAX, 1% penicillin-streptomycin, and 5 ng/ml basic fibroblast growth factor (bFGF, Peprotech). After one month of infection, the colonies were hand-picked and placed on feeder cells. The iPSCs were passed weekly through manual manipulation, with a daily medium replacement. When iPSCs were cultured without feeders, iPSCs were maintained in Essential 8 medium on vitronectin-coated dishes. These cells were passed using 0.5% EDTA in Dulbecco's phosphate-buffered saline (DPBS) every 5 days, while the medium was changed daily. Cell culture media, serum, and buffers were procured from Life Technologies (Grand Island, NY). Unless otherwise stated, the chemicals were procured from Sigma-Aldrich (St. Louis, MO).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The extraction of total cell RNA was performed using either QIAzol Lysis Reagent (QIAGEN).
Libraries for RNA sequencing were prepared using the Illumina Stranded mRNA Prep kit (#20040534) following the manufacturer’s protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
N11311_13
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| Data processing |
The quality of raw reads was assessed using FastQC (v0.11.9). Adaptor sequence trimming and poor read removal were performed using the cutadapt (v3.5).
The qualified reads were aligned to the human reference genome GRCh38 using the STAR (v2.7.8a) with the two-pass mode. Gene count was quantified based on the annotation of Gencode (v35)
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include read count values for each sample
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| Submission date |
May 13, 2024 |
| Last update date |
Aug 16, 2024 |
| Contact name |
Chia-Lang Hsu |
| E-mail(s) |
chialanghsu@ntuh.gov.tw
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| Organization name |
National Taiwan University Hospital
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| Department |
Department of Medical Research
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| Street address |
No. 7, Zhongshan S. Rd.
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| City |
Taipei |
| ZIP/Postal code |
100 |
| Country |
Taiwan |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE267287 |
Transcriptome profiling identified dysregulated signaling related to adipogenesis, metabolism, and inflammation in polycystic ovary syndrome patients |
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| Relations |
| BioSample |
SAMN41380052 |
| SRA |
SRX24533397 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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