2024-09-22
ENA first public
ERC000011
ENA-CHECKLIST
Junior Research Group Somatic Evolution and Early Detection German Cancer Research Center (DKFZ), Heidelberg, Germany
INSDC center name
public
INSDC status
ERS20609070
SRA accession
E-MTAB-13480_2:so91gc1
Submitter Id
3
month
age
ArrayExpress
broker name
granulosa cell
cell_type
not collected
collection date
house mouse
common name
Protocols: 3M old C57BL6/J mice were naturally or superovulated and COCs singularized. Granulosa cells were immediately flash frozen and matched oocytes were divided into individual drops of CARD media under mineral oil for fertilization. The oocytes were incubated in 37oC, 5% CO2 incubator for 30-40 min prior to fertilization. Frozen Mus musculus (C57BL6/J) male sperm straws were thawed and capacitated in Fertiup media for 30 min. Fertilization was achieved by combining 2.5 µl of activated sperm with 20 µl CARD media drop containing a single oocyte and incubated at 37oC, 5% CO2 for 3 hours. Then, oocytes were individually washed in Human Tubal Fluid (HTF) media drops and transferred to 20 µl HFT drops for overnight culture. After 24 hours, fertilization rate was evaluated by 2-cell stage and embryos transferred to G1+ media for further development. Early morula stage embryos were continuously collected based on observation 62-64 h post-fertilization. For late blastocysts, fertilization media was additionally changed to G2+ after 48h post-fertilization and blastocysts collected at 108h post-fertilization. For collection, the embryos were washed in DPBS and flash frozen.  Ovulation by hormonal stimulation was induced by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG - Pregmagon®) and second injection of 5 IU human chorionic gonadotropin (hCG - Ovogest®) 48 hours later. The COCs were collected from ampullas 14-16h after hCG injection. Granulosa cells and embryo samples were further processed by Smart-seq2, the cDNA amplification PCR cycle number for embryos was 16-17 and 20 for granulosa cells. Libraries were constructed using 1 ng of cDNA as template for library preparation, then tagmented, double indexed and amplified with 11 PCR cycles. All libraries were multiplexed into a single pool prior to sequencing.
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
SO91
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
female reproductive system
organism part
S
predicted group
Mus musculus
scientific_name
female
sex
superovulation
stimulus
C57BL/6J
strain
so91gc1
title